Quinacridone derivatives as labelling reagents for flurescence detection of bilogical materials

ABSTRACT

Disclosed are new quinacridone dye derivatives having characteristic fluorescence lifetimes. Also disclosed are methods for labelling target biological materials employing the quinacridone dyes and use of the labelled materials in biological assays. The quinacridone derivatives have the structure (I), in which Z 1  and Z 2  independently represent the atoms necessary to complete one ring, two fused ring, or three fused ring aromatic or heteroaromatic systems, each ring having five or six atoms selected from carbon atoms and optionally no more than two atoms selected from oxygen, nitrogen and sulphur; R 3 , R 4 , R 5 , R 6 , R 7  and R 8  are independently selected from hydrogen, halogen, amide, hydroxyl, cyano, nitro, mono- or di-nitro-substituted benzyl, amino, mono- or di-C 1 -C 4  alkyl-substituted amino, sulphydryl, carbonyl, carboxyl, C 1 -C 6  alkoxy, acrylate, vinyl, styryl, aryl, heteroaryl, C 1 -C 20  alkyl, aralkyl, sulphonate, sulphonic acid, quaternary ammonium, the group —E—F and the group —(CH 2 —) n Y; R 1  and R 2  are independently selected from hydrogen, mono- or di-nitro-substituted benzyl, C 1 -C 20  alkyl, aralkyl, the group —E—F and the group —(CH 2 —) n Y; E is a spacer group, F is a target bonding group; Y is selected from sulphonate, sulphate, phosphonate, phosphate, quaternary ammonium and carboxyl; and n is an integer from 1 to 6. The invention also relates to a set of different fluorescent quinacridone dye derivatives, each dye having a different fluorescence lifetime, the set of dyes being particularly useful for multiparameter analysis.

[0001] The present invention relates to new fluorescent labels. Inparticular the invention relates to new quinacridone derivatives thatcan be used as labels for attachment to target biological materials. Theinvention also relates to methods for labelling target biologicalmaterials and use of such labelled materials in biological assays.

[0002] There is an increasing interest in, and demand for, fluorescentlabels for use in the labelling and detection of biological materials.Fluorescent labels are generally stable, sensitive and a wide range ofmethods are now available for labelling biomolecules. Typically, theemission spectrum of a fluorescent dye is a characteristic property ofthe is dye. Measurements of the fluorescence intensity, the fluorescencelifetime, or fluorescence polarisation may be used in the detection andquantitation of materials labelled with that dye. One problem withmeasurements of fluorescence intensity as a means of detecting and/ormeasuring the concentration of a fluorescent labelled biomolecule isthat background fluorescence may interfere with the measurement. Thus,in order to obtain improvements in the sensitivity of fluorescencedetection, it is highly desirable to improve the signal-to-noise ratio.

[0003] One means of overcoming the problem of background noise has beenthrough the use of long wavelength dyes, for example, the cyanine dyesCy™5 and Cy7, as disclosed in U.S. Pat. No. 5,268,486 (Waggoner et al).These dyes emit in the 600-750 nm region of the spectrum, wherebackground fluorescence is much less of a problem. Another means ofimproving the signal-to-noise ratio in fluorescence measurements isthrough the use of time-resolved fluorescence, for example by usingfluorescent labels based on lanthanide chelates, eg. Eu³⁺ and Tb³⁺(Selvin et al, U.S. Pat. No. 562,282). In time-resolved fluorescentlabels, the fluorescence emission is typically longer than that of thebackground fluorescence, which may therefore be gated out usingappropriate instrumentation.

[0004] Linear trans-quinacridones are highly fluorescent andquinacridone derivatives have been developed as organic pigments (U.S.Pat. No. 2,844,484 (Reidinger, A. D. et al), U.S. Pat. No. 3,386,843(Jaffe, E. E. et al)), for use in high sensitivity photosensors andorganic light-emitting diodes and optical probes. Liu, P-H et al(J.Photochem.Photobiol., (2000), 137, 99-104) have synthesised a numberof 5,12-N,N′-dialkyl-2,9-dialkoxy quinacridones and have investigatedtheir spectral properties. Klein, G. et al (J.Chem.Soc.Chem.Commun.,(2001), 561-2) have prepared ethylenediamine functionalized quinacridonederivatives for use as fluorescent metal sensors.

[0005] Val'kova, G. et al (Dokl. Akad. Nauk. SSR, (1 978), 240(4),884-7) have measured the fluorescence lifetime of quinacridone, however,to date, there appear to be no reports relating to the use ofquinacridones as dyes suitable for labelling and the detection ofbiological materials such as nucleic acids, peptides, proteins,antibodies, drugs, hormones, cells and the like. The present inventiontherefore describes modifications of the quinacridone chromophore, toproduce a range of quinacridone derivatives which are useful forlabelling biological materials. The quinacridone derivatives of thepresent invention moreover provide a valuable set of fluorescent labelshaving a common core structure and which are particularly useful formultiparameter analysis. In each dye of a set of dyes, the absorptionand emission spectra remain essentially the same, whilst thefluorescence lifetimes of the dyes vary. Thus, it is possible to use acommon excitation source and determine the fluorescence lifetimes at thesame emission wavelength, thereby simplifying requirements for detectioninstrumentation used in multiparameter experiments. Another advantage ofthe dyes according to the present invention is that the fluorescenceemission wavelengths and lifetimes of the quinacridone derivatives aregenerally longer than the lifetimes of other fluorescent labels as wellas naturally occurring fluorescent materials, such as proteins andpolynucleotides, thereby allowing easy discrimination from backgroundfluorescence in assays utilising such dyes.

[0006] Accordingly in a first aspect, the present invention provides useof a reagent for labelling a target biological material, wherein saidreagent is a dye of formula (I):

[0007] wherein:

[0008] groups R³ and R⁴ are attached to the Z¹ ring structure and groupsR⁵ and R⁶ are attached to the Z² ring structure;

[0009] Z¹ and Z² independently represent the atoms necessary to completeone ring, two fused ring, or three fused ring aromatic or heteroaromaticsystems, each ring having five or six atoms selected from carbon atomsand optionally no more than two atoms selected from oxygen, nitrogen andsulphur;

[0010] R³, R⁴, R⁵, R⁶, R⁷ and R⁸ are independently selected fromhydrogen, halogen, amide, hydroxyl, cyano, nitro, mono- ordi-nitro-substituted benzyl, amino, mono- or di-C₁-C₄ alkyl-substitutedamino, sulphydryl, carbonyl, carboxyl, C₁-C₆ alkoxy, acrylate, vinyl,styryl, aryl, heteroaryl, C₁-C₂₀ alkyl, aralkyl, sulphonate, sulphonicacid, quaternary ammonium, the group —E—F and the group —(CH₂—)_(n)Y;

[0011] R¹ and R² are independently selected from hydrogen, mono- ordi-nitro-substituted benzyl, C₁-C₂₀ alkyl, aralkyl, the group —E—F andthe group —(CH₂—)_(n)Y;

[0012] E is a spacer group having a chain from 1-60 atoms selected fromthe group consisting of carbon, nitrogen, oxygen, sulphur and phosphorusatoms and F is a target bonding group;

[0013] Y is selected from sulphonate, sulphate, phosphonate, phosphate,quaternary ammonium and carboxyl; and n is an integer from 1 to 6.

[0014] In a first embodiment of the first aspect, the dye of formula (I)is a fluorescent dye wherein:

[0015] groups R³ and R⁴ are attached to atoms of the Z¹ ring structureand groups R⁵ and R⁶ are attached to atoms of the Z² ring structure,where Z¹ and Z² are hereinbefore defined;

[0016] R³, R⁴, R⁵, R⁶, R⁷ and R⁸ are independently selected fromhydrogen, halogen, amide, hydroxyl, cyano, amino, mono- or di-C₁-C₄alkyl-substituted amino, sulphydryl, carbonyl, carboxyl, C₁-C₆ alkoxy,acrylate, vinyl, styryl, aryl, heteroaryl, C₁-C₂₀ alkyl, aralkyl,sulphonate, sulphonic acid, quaternary ammonium, the group —E—F and thegroup —(CH₂—)_(n)Y; and

[0017] R¹ and R² are independently selected from hydrogen, C₁-C₂₀ alkyl,aralkyl, the group —E—F and the group —(CH₂—)_(n)Y;

[0018] wherein E, F, Y and n are hereinbefore defined.

[0019] The quinacridone dyes according to the first embodiment of thefirst aspect are particularly suitable for use as fluorescence lifetimedyes. In the context of the present invention, the term lifetime dye isintended to mean a dye having a measurable fluorescence lifetime,defined as the average amount of time that the dye remains in itsexcited state following excitation (Lakowicz, J. R., Principles ofFluorescence Spectroscopy, Kluwer Academic/Plenum Publishers, New York,(1999)). Alternatively, the dyes may be used in assays utilisingfluorescence polarisation.

[0020] Suitably, the fluorescent dyes according to the first embodimentof the first aspect exhibit a fluorescence lifetime in the range from 1to 30 nanoseconds. Preferably, the fluorescent lifetimes of the dyes arein the range from 10 to 25 nanoseconds.

[0021] In a second embodiment of the first aspect, the dye of formula(I) is a non-fluorescent or substantially non-fluorescent dye wherein:

[0022] groups R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, Z¹ and Z² are hereinbeforedefined and wherein at least one of groups R¹, R², R³, R⁴, R⁵, R⁶, R⁷and R⁸ comprises at least one nitro group.

[0023] In this embodiment, suitably, the at least one nitro group may beattached directly to the Z¹ and/or Z² ring structures. In thealternative, a mono- or di-nitro-substituted benzyl group may beattached to the R¹, R², R³, R⁴, R⁵, R⁶, R⁷ or R⁸ positions, whichoptionally may be further substituted with one or more nitro groupsattached directly to the Z¹ and/or Z² ring structures.

[0024] Preferably, in the first and second embodiments, at least one ofgroups R¹, R², R³, R⁴, R⁵, R⁶, R⁷ and R⁸ in the dye of formula (I) isthe group —E—F where E and F are hereinbefore defined.

[0025] Suitably, the target bonding group F is a reactive or functionalgroup. A reactive group of a dye of formula (I) can react under suitableconditions with a functional group of a target material; a functionalgroup of a dye of formula (I) can react under suitable conditions with areactive group of the target material such that the target materialbecomes labelled with the compound.

[0026] Preferably, when F is a reactive group, it is selected fromsuccinimidyl ester, sulpho-succinimidyl ester, isothiocyanate,maleimide, haloacetamide, acid halide, vinylsulphone, dichlorotriazine,carbodiimide, hydrazide and phosphoramidite. Preferably, when F is afunctional group, it is selected from hydroxy, amino, sulphydryl,imidazole, carbonyl including aldehyde and ketone, phosphate andthiophosphate. By virtue of these reactive and functional groups the dyeof formula (I) may react with and covalently bond to target materials.

[0027] Suitably, Z¹ and Z² may be selected from the group consisting ofphenyl, pyridinyl, naphthyl, anthranyl, indenyl, fluorenyl, quinolinyl,indolyl, benzothiophenyl, benzofuranyl and benzimidazolyl moieties.Additional one, two fused, or three fused ring structures will bereadily apparent to the skilled person. Preferred Z¹ and Z² are selectedfrom the group consisting of phenyl, pyridinyl, naphthyl, quinolinyl andindolyl moieties. Particularly preferred Z¹ and Z² are phenyl andnaphthyl moieties.

[0028] Preferably, at least one of the groups R¹, R², R³, R⁴, R⁵, R⁶, R⁷and R⁸ of the dyes of formula (I) is a water solubilising group forconferring a hydrophilic characteristic to the compound. Solubilisinggroups, for example, sulphonate, sulphonic acid and quaternary ammonium,may be attached directly to the aromatic ring structures Z¹ and/or Z² ofthe compound of formula (I). Alternatively, solubilising groups may beattached by means of a C₁ to C₆ alkyl linker chain to said aromatic ringstructures and may be selected from the group —(CH₂—)_(n)Y where Y isselected from sulphonate, sulphate, phosphonate, phosphate, quaternaryammonium and carboxyl; and n is an integer from 1 to 6. Alternativesolubilising groups may be carbohydrate residues, for example,monosaccharides. Examples of water solubilising constituents includeC₁-C₆ alkyl sulphonates, such as —(CH₂)₃—SO₃— and —(CH₂)₄—SO₃—. However,one or more sulphonate or sulphonic acid groups attached directly to thearomatic ring structures of a dye of formula (I) are particularlypreferred. Water solubility may be advantageous when labelling proteins.

[0029] Suitable spacer groups E may contain 1-60 chain atoms selectedfrom the group consisting of carbon, nitrogen, oxygen, sulphur andphosphorus. For example the spacer group may be:

[0030] —(CHR′)_(p)—

[0031] —{(CHR′)_(q)—O—(CHR′)_(r)}_(s)—

[0032] —{(CHR′)_(q)—NR′—(CHR′)_(r)}_(s)—

[0033] —{(CHR′)_(q)—(CH═CH)—(CHR′)_(r)}_(s)—

[0034] —{(CHR′)_(q)—Ar—(CHR′)_(r)}_(s)—

[0035] —{(CHR′)_(q)—CO—NR′—(CHR′)_(r)}_(s)—

[0036] —{(CHR′)_(q)—CO—Ar—NR′—(CHR′)_(r)}_(s)—

[0037] where R′ is hydrogen, C₁-C₄ alkyl or aryl, which may beoptionally substituted with sulphonate, Ar is phenylene, optionallysubstituted with sulphonate, p is 1-20, preferably 1-10, q is 0-10, r is1-10 and s is 1-5.

[0038] Specific examples of reactive groups R¹, R², R³, R⁴, R⁵, R⁶, R⁷and R⁸ and the groups with which R¹, R², R³, R⁴, R⁵, R⁶, R⁷ and R⁸ canreact are provided in Table 1. In the alternative, groups R¹, R², R³,R⁴, R⁵, R⁶, R⁷ and R⁸ may be the functional groups of Table 1 whichwould react with the reactive groups of a target material. TABLE 1Possible Reactive Substituents and Sites Reactive Therewith ReactiveGroups Functional Groups succinimidyl esters primary amino, secondaryamino isothiocyanates amino groups haloacetamides, maleimidessulphydryl, imidazole, hydroxyl, amine acid halides amino groupsanhydrides primary amino, secondary amino, hydroxyl hydrazides,aldehydes, ketones vinylsulphones amino groups dichlorotriazines aminogroups carbodiimides carboxyl groups phosphoramidites hydroxyl groups

[0039] Preferred reactive groups which are especially useful forlabelling target materials with available amino and hydroxyl functionalgroups include:

[0040] where n is 0 or an integer from 1-10.

[0041] Aryl is an aromatic substituent containing one or two fusedaromatic rings containing 6 to 10 carbon atoms, for example phenyl ornaphthyl, the aryl being optionally and independently substituted by oneor more substituents, for example halogen, hydroxyl, straight orbranched chain alkyl groups containing 1 to 10 carbon atoms, aralkyl andC₁-C₆ alkoxy, for example methoxy, ethoxy, propoxy and n-butoxy.

[0042] Heteroaryl is a mono- or bicyclic 5 to 10 membered aromatic ringsystem containing at least one and no more than 3 heteroatoms which maybe selected from N, O, and S and is optionally and independentlysubstituted by one or more substituents, for example halogen, hydroxyl,straight or branched chain alkyl groups containing 1 to 10 carbon atoms,aralkyl and C₁-C₆ alkoxy, for example methoxy, ethoxy, propoxy andn-butoxy.

[0043] Aralkyl is a C₁ to C₆ alkyl group substituted by an aryl orheteroaryl group.

[0044] Halogen and halo groups are selected from fluorine, chlorine,bromine and iodine.

[0045] Exemplary dyes according to the first embodiment of the firstaspect are as follows:

[0046] i)6-{2,9-dimethoxy-12-(5-carboxypentyl)-7,14-dioxo-7,14-dihydro-12H-quino[2,3-b]acridin-5-yl}hexanoicacid, diethyl ester;

[0047] ii)6-{2,9-dibromo-12-(5-carboxypentyl)-7,14-dioxo-7,14-dihydro-12H-quino[2,3-b]acridin-5-yl}hexanoicacid;

[0048] iii)6-{12-ethyl-7,14-dioxo-2,9-disulpho-7,14-dihydro-12H-quino[2,3-b]acridin-5-yl}hexanoicacid.

[0049] The fluorescent dyes according to the present invention may beused to label and thereby impart fluorescent properties to a variety oftarget biological materials. Thus, in a second aspect, there is provideda method for labelling a target biological material the methodcomprising:

[0050] i) adding to a liquid containing said target biological materiala dye of formula (I):

[0051] wherein:

[0052] groups R³ and R⁴ are attached to the Z¹ ring structure and groupsR⁵ and R⁶ are attached to the Z² ring structure, where Z¹ and Z² arehereinbefore defined;

[0053] R³, R⁴, R⁵, R⁶, R⁷ and R⁸ are independently selected fromhydrogen, halogen, amide, hydroxyl, cyano, amino, mono- or di-C₁-C₄alkyl-substituted amino, sulphydryl, carbonyl, carboxyl, C₁-C₆ alkoxy,acrylate, vinyl, styryl, aryl, heteroaryl, C₁-C₂₀ alkyl, aralkyl,sulphonate, sulphonic acid, quaternary ammonium, the group —E—F and thegroup —(CH₂—)_(n)Y;

[0054] R¹ and R² are independently selected from hydrogen, C₁-C₂₀ alkyl,aralkyl, the group —E—F and the group —(CH₂—)_(n)Y;

[0055] where E, F, Y and n are hereinbefore defined; and

[0056] ii) incubating said dye with said target biological materialunder conditions suitable for labelling said target.

[0057] Suitably, the fluorescent dyes of the present invention whereinat least one of the groups R¹ to R⁸ contains a charge, for example,quaternary amino, may be used to bind non-covalently to chargedbiological molecules such as, for example, DNA and RNA. Alternatively,fluorescent dyes of the present invention wherein at least one of thegroups R¹ to R⁸ is an uncharged group, for example, a long chain alkyl,an aryl group, or an ester group may be used to bind to and therebylabel uncharged biological molecules such as, for example, biologicallipids, as well as to intact cell membranes, membrane fragments andcells.

[0058] In a preferred embodiment according to the second aspect, atleast one of groups R¹, R², R³, R⁴, R⁵ , R⁶, R⁷ and R⁸ in the dye offormula (I) is the group —E—F where E and F are hereinbefore defined. Inthis embodiment, the fluorescent dyes may be used to covalently label atarget biological material. The target bonding group may be a reactivegroup for reacting with a functional group of the target material.Alternatively, the target bonding group may be a functional group forreacting with a reactive group on the target biological material. Themethod comprises incubating the target biological material with anamount of the dye according to the invention under conditions to form acovalent linkage between the target material and the dye. The target maybe incubated with an amount of a compound according to the presentinvention having at least one of groups R¹, R², R³, R⁴, R⁵, R⁶, R⁷ andR⁸ that includes a reactive or functional group that can covalently bindwith the functional or reactive group of the target biological material.

[0059] Suitable target biological materials include, but are not limitedto the group consisting of antibody, lipid, protein, peptide,carbohydrate, nucleotides which contain or are derivatized to containone or more of an amino, sulphydryl, carbonyl, hydroxyl and carboxyl,phosphate and thiophosphate groups, and oxy or deoxy polynucleic acidswhich contain or are derivatized to contain one or more of an amino,sulphydryl, carbonyl, hydroxyl and carboxyl, phosphate and thiophosphategroups, microbial materials, drugs, hormones, cells, cell membranes andtoxins.

[0060] Fluorescent dyes according to the present invention may be usedin assay methods that employ fluorescent labels for the detection and/ormeasurement of analytes using, for example, fluorescence intensity,fluorescence lifetime, or fluorescence polarisation measurements.Examples of such assays include protein-protein binding assays,immunoassays and nucleic acid hybridisation assays.

[0061] In a third aspect, there is provided a method for the assay of ananalyte in a sample which method comprises:

[0062] i) contacting the analyte with a specific binding partner forsaid analyte under conditions suitable to cause the binding of at leasta portion of said analyte to said specific binding partner to form acomplex and wherein one of said analyte and said specific bindingpartner is labelled with a fluorescent dye of formula (I):

[0063] wherein:

[0064] groups R³ and R⁴ are attached to atoms of the Z¹ ring structureand groups R⁵ and R⁶ are attached to atoms of the Z² ring structure,where Z¹ and Z² are hereinbefore defined;

[0065] at least one of groups R¹, R², R³, R⁴, R⁵, R⁶, R⁷ and R⁸ is thegroup —E—F where E is a spacer group having a chain from 1-60 atomsselected from the group consisting of carbon, nitrogen, oxygen, sulphurand phosphorus atoms and F is a target bonding group;

[0066] when any of said groups R³, R⁴, R⁵, R⁶, R⁷ and R⁸ is not saidgroup —E—F, said remaining groups R³, R⁴, R⁵, R⁶, R⁷ and R⁸ areindependently selected from hydrogen, halogen, amide, hydroxyl, cyano,amino, mono- or di-C₁-C₄ alkyl-substituted amino, sulphydryl, carbonyl,carboxyl, C₁-C₈ alkoxy, acrylate, vinyl, styryl, aryl, heteroaryl,C₁-C₂₀ alkyl, aralkyl, sulphonate, sulphonic acid, quaternary ammoniumand the group —(CH₂—)_(n)Y; and,

[0067] when any of groups R¹ and R² is not said group —E—F, saidremaining groups R¹ and R² are independently selected from hydrogen,C₁-C₂₀ alkyl, aralkyl and the group —(CH₂—)_(n)Y;

[0068] wherein Y and n are hereinbefore defined;

[0069] ii) measuring the emitted fluorescence of the labelled complex;and

[0070] iii) correlating the emitted fluorescence with the presence orthe amount of said analyte in said sample.

[0071] Suitably, step ii) may be performed by measurement of thefluorescence intensity, fluorescence lifetime, or fluorescencepolarisation of the labelled complex. Preferably, the measuring step ii)is performed by measuring the fluorescence lifetime, or the fluorescencepolarisation of the labelled complex.

[0072] In one embodiment, the assay method is a direct assay for themeasurement of an analyte in a sample. A known or putative inhibitorcompound may be optionally included in the reaction mixture.

[0073] In a second, or alternative embodiment, the assay may be acompetitive assay wherein a sample containing an analyte competes with afluorescent tracer for a limited number of binding sites on a bindingpartner that is capable of specifically binding the analyte and thetracer. Suitably, the tracer is a labelled analyte or a labelled analyteanalogue, in which the label is a fluorescent dye of formula (I).Increasing amounts (or concentrations) of the analyte in the sample willreduce the amount of the fluorescent labelled analyte or fluorescentlabelled analyte analogue that is bound to the specific binding partner.The fluorescence signal is measured and the concentration of analyte maybe obtained by interpolation from a standard curve.

[0074] In a further embodiment, the binding assay may employ a two-stepformat, wherein a first component (which may be optionally coupled to aninsoluble support) is bound to a second component to form a specificbinding complex, which is bound in turn to a third component. In thisformat, the third component is capable of specifically binding to eitherthe second component, or to the specific binding complex. Either of thesecond or the third component may be labelled with a fluorescent dyeaccording to the present invention. Examples include “sandwich” assays,in which one component of a specific binding pair, such as a firstantibody, is coated onto a surface, such as the wells of a multiwellplate. Following the binding of an antigen to the first antibody, afluorescent labelled second antibody is added to the assay mix, so as tobind with the antigen-first antibody complex. The fluorescence signal ismeasured and the concentration of antigen may be obtained byinterpolation from a standard curve.

[0075] In particularly preferred embodiments, the measurement step maybe performed using fluorescence polarisation. Thus, when the fluorescenttracer is not bound to the specific binding partner, it will tumble andreorientate rapidly relative to the fluorescence lifetime of thefluorescent dye. When bound to the specific binding partner, the tracerwill tumble and reorientate slowly relative to the fluorescence lifetimeof the dye. The degree of polarisation is therefore proportional to theextent of binding of the fluorescent tracer in the sample and inverselyproportional to the amount of analyte in the sample.

[0076] Examples of analyte-specific binding partner pairs include, butare not restricted to, antibodies/antigens, lectins/glycoproteins,biotin/streptavidin, hormone/receptor, enzyme/substrate or co-factor,DNA/DNA, DNA/RNA and DNA/binding protein. It is to be understood thatany molecules which possess a specific binding affinity for each othermay be employed, so that the fluorescent dyes of the present inventionmay be used for labelling one component of a specific binding pair,which in turn may be used in the detection of binding to the othercomponent.

[0077] The dyes according to the present invention may also be used inenzyme assays, utilising fluorescence polarisation measurements. Anassay for the detection of enzyme activity may be configured as follows.A reaction mixture is prepared by combining the enzyme and a fluorogenicsubstrate labelled with a fluorescent dye according to the presentinvention. A known or putative inhibitor compound may be optionallyincluded in the reaction mixture. The progress of the reaction may bemonitored by observing a change in fluorescence polarisation of thesample.

[0078] Thus, in a fourth aspect, there is provided an assay method forthe determination of an enzyme in a sample, the method comprising:

[0079] i) providing a substrate for the enzyme wherein the substrate islabelled with a fluorescent dye of formula (I):

[0080] wherein:

[0081] groups R³ and R⁴ are attached to atoms of the Z¹ ring structureand groups R⁵ and R⁶ are attached to atoms of the Z² ring structure,where Z¹ and Z² are hereinbefore defined;

[0082] at least one of groups R¹, R², R³, R⁴, R⁵, R⁶, R⁷ and R⁸ is thegroup —E—F where E is a spacer group having a chain from 1-60 atomsselected from the group consisting of carbon, nitrogen, oxygen, sulphurand phosphorus atoms and F is a target bonding group;

[0083] when any of said groups R³, R⁴, R⁵, R⁶, R⁷ and R⁸ is not saidgroup —E—F, said remaining groups R³, R⁴, R⁵, R⁶, R⁷ and R⁸ areindependently selected from hydrogen, halogen, amide, hydroxyl, cyano,amino, mono- or di-C₁-C₄ alkyl-substituted amino, sulphydryl, carbonyl,carboxyl, C₁-C₆ alkoxy, acrylate, vinyl, styryl, aryl, heteroaryl,C₁-C₂₀ alkyl, aralkyl, sulphonate, sulphonic acid, quaternary ammoniumand the group —(CH₂—)_(n)Y; and, when any of groups R¹ and R² is notsaid group —E—F, said remaining groups Rand R² are independentlyselected from hydrogen, C₁-C₂₀ alkyl, aralkyl and the group—(CH₂—)_(n)Y;

[0084] wherein Y and n are hereinbefore defined;

[0085] ii) combining the labelled substrate with the enzyme underconditions suitable for initiating the enzymatic reaction; and

[0086] iii) measuring the fluorescence polarisation of the sample todetermine the extent of reaction.

[0087] Suitably, the enzyme may be selected from cleavage enzymes suchas proteases that catalyse cleavage of the substrate into two or morefragments, thereby resulting in a decrease in fluorescence polarisation.Alternatively the enzyme may join two components, for example, a ligaseor a transferase, resulting in an increase in polarisation of thesample.

[0088] The fluorescent dyes according to the first embodiment of thefirst aspect may be used in applications that include detecting anddistinguishing between various components in a mixture. In a fifthaspect, the present invention provides a set of two or more differentfluorescent dyes, each dye of said set of dyes having the formula (I):

[0089] wherein:

[0090] groups R³ and R⁴ are attached to atoms of the Z¹ ring structureand groups R⁵ and R⁶ are attached to atoms of the Z² ring structure,where Z¹ and Z² are hereinbefore defined;

[0091] R³, R⁴, R⁵, R⁶, R⁷ and R⁸ are independently selected fromhydrogen, halogen, amide, hydroxyl, cyano, amino, mono- or di-C₁-C₄alkyl-substituted amino, sulphydryl, carbonyl, carboxyl, C₁-C₆ alkoxy,acrylate, vinyl, styryl, aryl, heteroaryl, C₁-C₂₀ alkyl, aralkyl,sulphonate, sulphonic acid, quaternary ammonium, the group —E—F and thegroup —(CH₂—)_(n)Y;

[0092] R¹ and R² are independently selected from hydrogen, C₁-C₂₀ alkyl,aralkyl, the group —E—F and the group —(CH₂—)_(n)Y;

[0093] E is a spacer group having a chain from 1-60 atoms selected fromthe group consisting of carbon, nitrogen, oxygen, sulphur and phosphorusatoms and F is a target bonding group;

[0094] Y is selected from sulphonate, sulphate, phosphonate, phosphate,quaternary ammonium and carboxyl; and n is an integer from 1 to 6;

[0095] wherein each dye of said set has a distinguishably differentfluorescence lifetime compared with the lifetimes of the remaining dyesof the set.

[0096] Preferably, in each dye of the set of dyes at least one of groupsR¹, R², R³, R⁴, R⁵, R⁶, R⁷ and R⁸ is the group —E—F where E and F arehereinbefore defined.

[0097] Preferably, the set of fluorescent dyes according to theinvention will comprise four different dyes, each dye of the set havinga different fluorescence lifetime. Preferably, each of the fluorescentdyes of the set of dyes exhibits a fluorescence lifetime in the rangefrom 1 to 30 nanoseconds, more preferably, in the range from 10 to 25nanoseconds.

[0098] To distinguish between different fluorescent dyes in the set ofdyes, the lifetime of the fluorescence emission of each of the dyes ispreferably separated by at least 0.5 nanoseconds.

[0099] The set of dyes may be used in a detection method whereindifferent fluorescent dyes of the set of dyes are covalently bonded to aplurality of different primary components, each primary component beingspecific for a different secondary component, in order to identify eachof a plurality of secondary components in a mixture of secondarycomponents. The method comprises covalently binding different dyes of aset of fluorescent dyes according to the fifth aspect of the inventionto different primary components in a multicomponent mixture wherein eachdye of the set has a different fluorescence lifetime, compared with thefluorescence lifetimes of the remaining dyes of the set; adding thedye-labelled primary components to a preparation containing secondarycomponents under conditions to enable binding of at least a portion ofeach of said dye-labelled primary components to its respective secondarycomponent; and determining the presence or the amount of the boundsecondary component by measuring the fluorescence lifetime of each ofthe labelled primary component-secondary component complexes.

[0100] If required, any unreacted primary components may be removed orseparated from the preparation by, for example washing, to preventinterference with the analysis.

[0101] Preferably, a single wavelength of excitation can be used toexcite fluorescence from two or more materials in a mixture, where eachfluoresces having a different characteristic fluorescent lifetime.

[0102] The set of fluorescent dyes according to the present inventionmay be used in any system in which the creation of a fluorescent primarycomponent is possible. For example, an appropriately reactivefluorescent dye according to the invention can be conjugated to a DNA orRNA fragment and the resultant conjugate then caused to bind to acomplementary target strand of DNA or RNA. Other examples of primarycomponent-secondary component complexes which may be detected includeantibodies/antigens and biotin/streptavidin.

[0103] The set of fluorescent dyes according to the present inventionmay also be advantageously used in fluorescent DNA sequencing based uponfluorescence lifetime discrimination of the DNA fragments. Briefly, eachone of a set of dyes, may be coupled to a primer. Various primers areavailable, such as primers from pUC/M13, λgt10, λgt11 and the like (seeSambrook et al, Molecular Cloning, A Laboratory Manual 2^(nd) Edition,Cold Spring Harbour Laboratory Press 1989). DNA sequences are clonedinto an appropriate vector having a primer sequence joined to the DNAfragment to be sequenced. After hybridisation to the DNA template,polymerase enzyme-directed synthesis of a complementary strand occurs.Different 2′,3′-dideoxynucleotide terminators are employed in each isdifferent sequencing reaction so as to obtain base-specific terminationof the chain extension reaction. The resulting set of DNA fragments areseparated by electrophoresis and the terminating nucleotide (and thusthe DNA sequence) is determined by detecting the fluorescence lifetimeof the labelled fragments. DNA sequencing may also be performed usingdideoxynucleotide terminators covalently labelled with the fluorescentdyes according to the present invention.

[0104] The non-fluorescent or substantially non-fluorescent dyesaccording to the second embodiment of the first aspect may be used asthe substrate for an enzyme and which upon reaction with the enzyme,yields a fluorescent product.

[0105] Bacterial nitroreductases have been shown to catalyse the generalreaction set out below in Reaction Scheme 1.

[0106] where, in the presence of NADH or NADPH, one or more nitro groupson an organic molecule may be reduced to a hydroxylamine (—NHOH) groupwhich may subsequently be converted to an amine (—NH₂) group.

[0107] Thus, in a sixth aspect of the invention, there is provided amethod of increasing the fluorescence of a non-fluorescent orsubstantially non-fluorescent dye of formula (I):

[0108] wherein:

[0109] groups R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, Z¹ and Z² are hereinbeforedefined; and wherein at least one of groups R¹, R², R³, R⁴, R⁵, R⁶, R⁷and R⁸ comprises at least one nitro group;

[0110] characterised by the reduction of said at least one nitro groupto —NHOH or —NH₂

[0111] Suitably, reduction is by means of nitroreductase. This can beachieved by enzymatic conversion of a nitro group in a compound offormula (I) to a —NHOH or —NH₂ group by the action of thenitroreductase. Depending on the structure of the dye, the intensityand/or lifetime of the fluorescence emission from the product of thenitroreductase reaction may be increased so as to exhibit a lifetimetypically in the range from 1 to 30 nanoseconds. Moreover, thefluorescence lifetime characteristics of the reaction product can bealtered to suit the application by means of additional substitutents,whilst retaining the nitro group(s) that are involved in the reactionwith nitroreductase. Thus, fluorescent reporters compatible for use withother fluors in multiplex systems can be provided.

[0112] In a seventh aspect of the invention there is provided a methodfor detecting nitroreductase enzyme activity in a compositioncomprising:

[0113] i) mixing said composition under conditions to promotenitroreductase activity with a dye of formula (I):

[0114] wherein:

[0115] groups R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, Z¹ and Z² are hereinbeforedefined and wherein at least one of groups R¹, R², R³, R⁴, R⁵, R⁶, R⁷and R⁸ comprises at least one nitro group; and

[0116] ii) measuring an increase in fluorescence wherein said increaseis a measure of the amount of nitroreductase activity.

[0117] Suitably, the measurement step (ii) may be a measure offluorescence intensity and/or fluorescence lifetime of the labelledproduct of the nitroreductase reaction.

[0118] In one embodiment of the seventh aspect, the compositioncomprises a cell or cell extract. In principle, any type of cell can beused, i.e. prokaryotic or eukaryotic (including bacterial, mammalian andplant cells). Where appropriate, a cell extract can be prepared from acell, using standard methods known to those skilled in the art(Molecular Cloning, A Laboratory Manual 2^(nd) Edition (1989), ColdSpring Harbour Laboratory Press), prior to measuring fluorescence.

[0119] Typical conditions for nitroreductase activity compriseincubation of the composition in a suitable medium and the dye atapproximately 37° C. in the presence of NADH and FMN.

[0120] In a eighth aspect of the invention there is provided an assaymethod comprising:

[0121] i) binding one component of a specific binding pair to a surface;

[0122] ii) adding a second component of the specific binding pair underconditions to promote binding between the components, said secondcomponent being labelled with a nitroreductase enzyme;

[0123] iii) adding a dye of formula (I):

[0124] wherein:

[0125] groups R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, Z¹ and Z² are hereinbeforedefined and wherein at least one of groups R¹, R², R³, R⁴, R⁵, R⁶, R⁷and R⁸ comprises at least one nitro group; and

[0126] iv) detecting binding of the second component to the firstcomponent by measuring an increase in fluorescence as a measure of boundnitroreductase activity.

[0127] In a preferred embodiment of the eighth aspect, said specificbinding pair is selected from the group consisting ofantibodies/antigens, lectins/glycoproteins, biotin/streptavidin,hormone/receptor, enzyme/substrate, DNA/DNA, DNA/RNA and DNA/bindingprotein.

[0128] Briefly, an in vitro assay method for the detection of antibodybinding may be configured as follows. An antibody specific for anantigen of interest may be labelled by covalently linking it to anenzymatically active nitroreductase. The labelled antibody can then beintroduced into the test sample containing the antigen under bindingconditions. After washing to remove any unbound antibody, the amount ofbound antibody is detected by incubating the sample with a substratecomprising a compound of formula (I) having at least one nitro groupunder conditions promoting nitroreductase activity and measuring anincrease in fluorescence. The amount of fluorescence detected will beproportional to the amount of nitroreductase-labelled antibody that hasbound to the analyte.

[0129] In an in vitro assay for detecting the binding of nucleic acidsby hybridisation, either of the pair of target and probe nucleic acid isbound to a membrane or surface. The unbound partner is labelled withnitroreductase and incubated under hybridising conditions with the boundnucleic acid. Unbound, labelled nucleic acid is washed off and theamount of bound, labelled nucleic acid is measured by incubating themembrane or surface with a compound of formula (I) having at least onenitro group under conditions suitable for nitroreductase activity. Theamount of increase in fluorescence gives a measure of the amount ofbound labelled DNA.

[0130] Methods for coupling enzymes, such as nitroreductase, to otherbiomolecules, e.g. proteins and nucleic acids, are well known(Bioconjugate Techniques, Academic Press 1996). Coupling may be achievedby direct means, for example by use of a suitable bifunctionalcrosslinking agent (e.g. N-[γ-maleimidopropionic acid]hydrazine, Pierce)to covalently link the enzyme and binding partner. Alternatively,coupling may be achieved by indirect means, for example by separatelybiotinylating the enzyme and the binding partner using a chemicallyreactive biotin derivative, (e.g. N-hydroxysuccinimido-biotin, Pierce)and subsequently coupling the molecules through a streptavidin bridgingmolecule.

[0131] Cell based assays are increasingly attractive over in vitrobiochemical assays for use in high throughput screening (HTS). This isbecause cell based assays require minimal manipulation and the readoutscan be examined in a biological context that more faithfully mimics thenormal physiological situation. Such in vivo assays require an abilityto measure a cellular process and a means to measure its output. Forexample, a change in the pattern of transcription of a number of genescan be induced by cellular signals triggered, for example, by theinteraction of an agonist with its cell surface receptor or by internalcellular events such as DNA damage. The induced changes in transcriptioncan be identified by fusing a reporter gene to a promoter region whichis known to be responsive to the specific activation signal.

[0132] In fluorescence-based enzyme-substrate systems, an increase influorescence gives a measure of the activation of the expression of thereporter gene.

[0133] Accordingly, in a ninth aspect of the invention, there isprovided an assay method which comprises:

[0134] i) contacting a host cell which has been transfected with anucleic acid molecule comprising expression control sequences operablylinked to a sequence encoding a nitroreductase with a dye of formula(I):

[0135] wherein:

[0136] groups R¹, R², R³ ₁, R⁴, R⁵, R⁶ ₁, R⁷, R⁸, Z¹ and Z² arehereinbefore defined and wherein at least one of groups R¹, R², R³, R⁴,R⁵, R⁶, R⁷ and R⁸ comprises at least one nitro group; and

[0137] ii) measuring an increase in fluorescence as a measure ofnitroreductase gene expression.

[0138] Suitably, the measurement step (ii) may be a measure offluorescence intensity and/or fluorescence lifetime of the labelledproduct of the nitroreductase reaction.

[0139] In one embodiment of the ninth aspect, the assay method isconducted in the presence of a test agent whose effect on geneexpression is to be determined.

[0140] Methods for using a variety of enzyme genes as reporter genes inmammalian cells are well known (for review see Naylor L. H., BiochemicalPharmacology, (1999), 58, 749-757). The reporter gene is chosen to allowthe product of the gene to be measurable in the presence of othercellular proteins and is introduced into the cell under the control of achosen regulatory sequence which is responsive to changes in geneexpression in the host cell. Typical regulatory sequences include thoseresponsive to hormones, second messengers and other cellular control andsignalling factors. For example, agonist binding to seven transmembranereceptors is known to modulate promoter elements including the cAMPresponsive element, NF-AT, SRE and AP1; MAP kinase activation leads tomodulation of SRE leading to Fos and Jun transcription; DNA damage leadsto activation of transcription of DNA repair enzymes and the tumoursuppressor gene p53. By selection of an appropriate regulatory sequencethe reporter gene can be used to assay the effect of added agents oncellular processes involving the chosen regulatory sequence under study.

[0141] For use as a reporter gene, the nitroreductase gene may beisolated by well known methods, for example by amplification from a cDNAlibrary by use of the polymerase chain reaction (PCR) (MolecularCloning, A Laboratory Manual 2^(nd) Edition, Cold Spring HarbourLaboratory Press (1989), pp 14.5-14.20). Once isolated, thenitroreductase gene may be inserted into a vector suitable for use withmammalian promoters (Molecular Cloning, A Laboratory Manual 2^(nd)Edition, Cold Spring Harbour Laboratory Press (1989), pp 16.56-16.57) inconjunction with and under the control of the gene regulatory sequenceunder study. The vector containing the nitroreductase reporter andassociated regulatory sequences may then be introduced into the hostcell by transfection using well known techniques, for example by use ofDEAE-Dextran or Calcium Phosphate (Molecular Cloning, A LaboratoryManual 2^(nd) Edition, Cold Spring Harbour Laboratory Press (1989), pp16.30-16.46). Other suitable techniques will be well known to thoseskilled in the art.

[0142] In another embodiment of the ninth aspect, the dye of formula (I)wherein groups R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, Z¹ and Z² arehereinbefore defined and wherein at least one of groups R¹, R², R³, R⁴,R⁵, R⁶, R⁷ and R⁸ comprises at least one nitro group, is permeable tocells. In this embodiment, preferably, at least one of groups R¹, R²,R³, R⁴, R⁵, R⁶, R⁷ and R⁸ comprises a cell membrane permeabilisinggroup. Membrane permeant compounds can be generated by maskinghydrophilic groups to provide more hydrophobic compounds. The maskinggroups can be designed to be cleaved from the substrate within the cellto generate the derived fluorogenic substrate intracellularly. Becausethe substrate is more hydrophilic than the membrane permeant derivative,it is then trapped in the cell. Suitable cell membrane permeabilisinggroups may be selected from acetoxymethyl ester, which is readilycleaved by endogenous mammalian intracellular esterases (Jansen, A. B.A. and Russell, T. J., J.Chem.Soc., (1965), 2127-2132 and Daehne W. etal. J.Med.Chem., (1970) 13, 697-612) and pivaloyl ester (Madhu et al.,J. Ocul.Pharmacol.Ther., (1998), 14(5), 389-399) although other suitablegroups will be recognised by those skilled in the art.

[0143] Typically, to assay the activity of a test agent to activatecellular responses via the regulatory sequence under study, cellstransfected with the nitroreductase reporter are incubated with the testagent, followed by addition of a dye of formula (I) wherein at least oneof groups R¹, R², R³, R⁴ and R⁵ in said dye comprises at least one nitrogroup, said compound being made cell permeant. After an appropriateperiod required for conversion of the substrate to a form exhibitingfluorescence characteristics, the fluorescence intensity and/orfluorescence lifetime from the cells is measured at an emissionwavelength appropriate for the chosen dye. Measurement of fluorescencemay be readily achieved by use of a range of detection instrumentsincluding fluorescence microscopes (e.g. LSM 410, Zeiss), microplatereaders (e.g. CytoFluor 4000, Perkin Elmer), CCD imaging systems (e.g.LEADseeker™, Amersham Pharmacia Biotech) and Flow Cytometers (e.g.FACScalibur, Becton Dickinson).

[0144] The measured fluorescence is compared with fluorescence fromcontrol cells not exposed to the test agent and the effects, if any, ofthe test agent on gene expression modulated through the regulatorysequence, is determined from the ratio of fluorescence in the test cellsto the fluorescence in the control cells. Where appropriate, a cellextract can be prepared using conventional methods.

[0145] Suitable means for expressing a nitroreductase enzyme include anexpression plasmid or other expression construct. Methods for preparingsuch expression constructs are well known to those skilled in the art.

[0146] In a tenth aspect of the present invention, there is provided adye of formula (I):

[0147] wherein:

[0148] groups R³ and R⁴ are attached to atoms of the Z¹ ring structureand groups R⁵ and R⁶ are attached to atoms of the Z² ring structure;

[0149] Z¹ and Z² independently represent the atoms necessary to completeone ring, two fused ring, or three fused ring aromatic or heteroaromaticsystems, each ring having five or six atoms selected from carbon atomsand optionally no more than two atoms selected from oxygen, nitrogen andsulphur;

[0150] at least one of groups R¹, R², R³, R⁴, R⁵, R⁶, R⁷ and R⁸ is thegroup —E—F where E is a spacer group having a chain from 1-60 atomsselected from the group consisting of carbon, nitrogen, oxygen, sulphurand phosphorus atoms and F is a target bonding group; and,

[0151] when any of said groups R³, R⁴, R⁵, R⁶, R⁷ and R⁸ is not saidgroup —E—F, said remaining groups R³, R⁴, R⁵, R⁶, R⁷ and R⁸ areindependently selected from hydrogen, halogen, amide, hydroxyl, cyano,nitro, amino, mono- or di-C₁-C₄ alkyl-substituted amino, sulphydryl,carbonyl, carboxyl, C₁-C₆ alkoxy, acrylate, vinyl, styryl, aryl,heteroaryl, C₁-C₂₀ alkyl, aralkyl, sulphonate, sulphonic acid,quaternary ammonium and the group —(CH₂—)_(n)Y; and,

[0152] when any of groups R¹ and R² is not said group —E—F, saidremaining groups R¹ and R² are independently selected from hydrogen,mono- or di-nitro-substituted benzyl, C₁-C₂₀ alkyl, aralkyl and thegroup —(CH₂—)_(n)Y;

[0153] E is a spacer group having a chain from 1-60 atoms selected fromthe group consisting of carbon, nitrogen, oxygen, sulphur and phosphorusatoms and F is a target bonding group;

[0154] Y is selected from sulphonate, sulphate, phosphonate, phosphate,quaternary ammonium and carboxyl; and n is an integer from 1 to 6;

[0155] provided that at least one of groups R¹, R², R³, R⁴, R⁵, R⁶, R⁷and R⁸ is a water solubilising group.

[0156] Preferably, the target bonding group F comprises a reactive groupfor reacting with a functional group on a target material, or afunctional group for reacting with a reactive group on a targetmaterial. Preferred reactive groups may be selected from carboxyl,succinimidyl ester, sulpho-succinimidyl ester, isothiocyanate,maleimide, haloacetamide, acid halide, hydrazide, vinylsulphone,dichlorotriazine and phosphoramidite. Preferred functional groups may beselected from hydroxy, amino, sulphydryl, imidazole, carbonyl includingaldehyde and ketone, phosphate and thiophosphate.

[0157] Preferably, the spacer group E is selected from:

[0158] —(CHR′)_(p)—

[0159] —{(CHR′)_(q)—O—(CHR′)_(r)}_(s)—

[0160] —{(CHR′)_(q)—NR′—(CHR′)_(r)}_(s)—

[0161] —{(CHR′)_(q)—(CH═CH)—(CHR′)_(r)}_(s)—

[0162] —{(CHR′)_(q)—Ar—(CHR′)_(r)}_(s)—

[0163] —{(CHR′)_(q)—CO—NR′—(CHR′)_(r)}_(s)—

[0164] —{(CHR′)_(q)—CO—Ar—NR′—(CHR′)_(r)}_(s)—

[0165] where R′ is hydrogen, C₁-C₄ alkyl or aryl, which may beoptionally substituted with sulphonate, Ar is phenylene, optionallysubstituted with sulphonate, p is 1-20, preferably 1-10, q is 0-10, r is1-10 and s is 1-5.

[0166] Dyes according to the tenth aspect may contain a polymerizablegroup suitable for the formation of a polymer containing the dye.Suitable polymerizable groups are selected from acrylate, methacrylateand acrylamide. Polymerization may be carried out with a suitablyderivatized compound of this invention used in conjunction with a secondpolymerizable monomer starting material, such as styrene orvinyltoluene, to form a copolymer containing the dye. The dyes of thepresent invention need not have a polymerisable group, for example, thedye may be incorporated during polymerisation or particle formation ormay be absorbed into or onto polymer particles.

[0167] The dyes of formula (I) may be prepared by a process comprisingreacting diethyl succinyl-succinate with an appropriately substitutedaniline according to published methods (see Jaffe, E. E. and Marshall,W. J., U.S. Pat. No. 3,386,843; Jaffe, E. E. and Ehrich, F. F. U.S. Pat.No. 3,873,548). For example, heating diethyl succinyl-succinate with4-aminobenzoic acid affordsdiethyl-2,5-di(4-carboxyanilino)-3,6-dihydroterphthalate. Furtherheating in a high boiling solvent affords2,9-dicarboxy-6,13-dihydroquinacridone. Oxidation with sodium3-nitrobenzene sulphonate gives 2,9-dicarboxyquinacridone. Alternativemethods of synthesising quinacridone and its derivatives are disclosedby Bender, H. et al (U.S. Pat. No. 4,956,464), whereby2,5-dianilino-3,6-dihydroterephthalic acid derivatives may be cyclizedand dehydrogenated at 500-600° C. Maki, H. et al (U.S. Pat. No.5,659,036) describe methods for preparing quinacridone derivatives inwhich alkyl esters of 1,4-cyclohexadione-2,5-dicarboxylic acid may bereacted with an appropriately substituted aromatic amine and theresultant 2,5-di(arylamino)-3,6-dihydroterephthalic acid dialkyl esterderivative may be cyclized to a 6,13-dihydroquinacridone which is thenoxidized with a nitrobenzene sulphonic acid to give the quinacridone.

[0168] It will be readily appreciated that certain dyes of formula (I)may be useful as intermediates for conversion to other dyes of formula(I) by methods well known to those skilled in the art. Likewise, certainof the intermediates may be useful for the synthesis of dyes of formula(I). The compounds of the present invention may be synthesized by themethods disclosed herein. Derivatives of the dyes having a particularutility are prepared either by selecting appropriate precursors or bymodifying the resultant compounds by known methods to include functionalgroups at a variety of positions. As examples, the dyes of the presentinvention may be modified to include certain reactive groups forpreparing a fluorescent labelling reagent, or charged or polar groupsmay be added to enhance the solubility of the compound in polar ornon-polar solvents or materials. As examples of conversions an ester maybe converted to a carboxylic acid or may be converted to an amidoderivative. Groups R¹ to R⁸ may be chosen so that the compounds of thepresent invention have different fluorescence characteristics, therebyproviding a number of related dyes which can be used in multiparameteranalyses wherein the presence and quantity of different compounds in asingle sample may be differentiated based on the wavelengths andlifetimes of a number of detected fluorescence emissions. The dyes ofthe present invention may be made soluble in aqueous, other polar, ornon-polar media containing the material to be labelled by appropriateselection of R-groups.

[0169] The invention is further illustrated by reference to thefollowing examples and figures in which:

[0170]FIG. 1 is a plot showing the absorbance and emission spectrum of6-{7,14-dioxo-2,9-disulpho-7,14-dihydro-12H-quino[2,3-b]acridin-5-yl}hexanoicacid;

[0171]FIG. 2 shows the fluorescence lifetime decay plots of three dyesaccording to the present invention;

[0172]FIG. 3 is a lifetime decay plot of6-{7,14-dioxo-2,9-disulpho-7,14-dihydro-12H-quino[2,3-b]acridin-5-yl)hexanoicacid and of its conjugate with ovalbumin;

[0173]FIG. 4 is a plot showing trypsin cleavage of a conjugate ofalbumin with6-{12-ethyl-7,14-dioxo-2,9-disulpho-7,14-dihydro-12H-quino[2,3-b]acridin-5-yl}hexanoicacid monitored by fluorescence polarisation.

[0174] Cy™ and LEADseeker™ are trademarks of Amersham Pharmacia BiotechUK Limited.

Examples

[0175] 1. 2-Bromo-5,12-dihexylquinacridone (A) and2,9-dibromo-5,12-dihexylquinacridone (B)

[0176] 1.1 5,12-Dihexylquinacridone

[0177] A 25 ml RB flask was charged with quinacridone (Dojindo;156 mg,0.5 mmol) and anhydrous N,N-dimethylformamide (5 ml). The flask waspurged with nitrogen and set stirring. To the mixture was added sodiumhydride (60 wt % dispersion in oil, 48 mg, 1.2 mmol) and the mixtureleft to stir. A blue colour slowly developed in the liquid phase butmuch of the solids remained undissolved after 2 hrs; dissolution wasachieved by the addition of dimethyl sulphoxide (3 ml) to give a deepblue solution after a further 1 hr. At this point 1-iodohexane (295 μl,2.0 mmol) was added and the mixture left to stir for 3 days. During thistime the blue colour was completely discharged and an orange solidprecipitated.

[0178] The mixture was quenched into 0.5M aqueous hydrochloric acid, thesolid collected by filtration and washed with excess water. The dampsolid was then dried by dissolution into dichloromethane containinganhydrous magnesium sulfate; the slurry was filtered and thebrown-orange filtrate evaporated under vacuum to give the crude product.This was purified by flash chromatography (silica. 0-2.5% ethyl acetatein dichloromethane); pure fractions were pooled, filtered andevaporated, then triturated with diethyl ether to give an orange powder.Yield=161 mg (67%). λ_(max) (CH₂Cl₂)=526, 493 nm. δ_(H) (200 MHz, CDCl₃)0.95 (6H, t), 1.20-1.65 (12H, m), 2.01 (4H, m), 4.52 (4H, app t), 7.28(2H, app t), 7.52 (2H, app d), 7.76 (2H, td), 8.58 (2H, dd) and 8.79(2H, s).

[0179] Mass spectrum: (ES+) 481 (M+H), 503 (M+Na). Accurate mass:(M+H)=C₃₂H₃₇N₂O₂, requires 481.2855. Found 481.2844 (−2.3 ppm).

[0180] 1.2 2-Bromo-5,12-dihexylquinacridone and2,9-dibromo-5,12-dihexylquinacridone

[0181] 5,12-Dihexylquinacridone (60 mg, 125 μmol) was mixed with ethanol(2.5 ml) and benzyltrimethylammonium tribromide (97 mg, 250 μmol). Theresulting slurry was stirred at ambient temperature for 24 hrs, thenheated under reflux for 16 hrs to give limited reaction. Addition ofchloroform (2.5 ml) dissolved all solids to give an orange solution,continued reflux gave moderate generation of mono- and di-brominatedproducts. After evaporation of solvent the products were isolated byflash chromatography (silica, dichloromethane) to give pure samples.

[0182]2-Bromo-5,12-dihexylquinacridone (A): λ_(max) (CH₂Cl₂)=526, 493nm. δ_(H) (200 MHz, CDCl₃) 0.95 (6H, m), 1.3-1.7 (12H, m), 1.9-2.1 (4H,m), 4.4-4.5 (4H, m), 7.25 (1H, m), 7.36 (1H, d), 7.48 (1H, d), 7.70-7.80(2H, m), 8.52 (1H, dd), 8.61 (1H, s), 8.67 (1H, s) and 8.71 (1H, s).Mass spectrum (DEI+): 558+560 (ratio 1:1) (M+).

[0183] 2,9-Dibromo-5,12-dihexylquinacridone (B): λ_(max) (CH₂Cl₂)=532,498 nm. δ_(H) (200 MHz, CDCl₃) 0.95 (6H, t), 1.3-1.7 (12H, m), 1.8-2.0(4H, m), 4.5 (4H, t), 7.36 (2H, d), 7.78 (2H, dd), 8.58 (2H, d) and 8.64(2H, s). Mass spectrum (DEI+): 636+638+640 (ratio 1:2:1) (M+).

[0184] 2. Quinacridone-2,9-disulphonic acid, (di-potassium salt)

[0185] Quinacridone (200 mg, 0.645 mM) was placed in a round bottomedflask fitted with a magnetic stirrer and air condenser. The solid wasdissolved in 98% sulphuric acid (2 ml) and heated to 110° C. for 6 hrunder a nitrogen atmosphere. TLC (RP C-18 50/50 methanol/water) showedthat all the starting material had been converted to a single fastmoving component (visualised under long wavelength uv light). Thereaction was dripped onto 10 ml ice to give a dark red solution. Thiswas neutralised with solid potassium hydrogen carbonate to give a darkred precipitate. This was collected by centrifugation and thesupernatant discarded.

[0186] Recrystallisation from water gave 0.21 g (0.386 Mm, 60%) of redsolid identified as the di-potassium salt ofquinacridone-2,9-disulphonic acid. Mass Spec(ES+). MH⁺ 473.1; MK₂ ⁺550.5. MPt >300° C. λ_(max)(ab) 499, 527 nm (water); λ_(max)(em) 560,590nm (water).

[0187] 3. Quinacridone-2,4,9,11-tetrasulphonic acid (tetra-potassiumsalt)

[0188] Quinacridone (500 mg, 1.61 mM) was placed in a round bottomedflask fitted with a magnetic stirrer and air condenser. The solid wasdissolved in 20% oleum (5 mL) and heated to 110° C. for 20 hr under anitrogen atmosphere. TLC (RP C-18 50/50 methanol/water) showed that allthe starting material had been converted to a major fast movingcomponent with two minor faster moving and one minor slower movingcomponent (visualised under long wavelength uv light). The reaction wasdripped onto 10 ml ice to give a dark red solution. This was neutralisedwith solid potassium hydrogen carbonate to give an orange precipitate.This was collected by centrifugation and the supernatant discarded.

[0189] Recrystallisation twice from water gave 1.1 gm of an orange solididentified as the tetra-potassium salt ofquinacridone-2,4,9,11-tetrasulphonic acid. Mass Spec (ES+). MH⁺ 632.9.

[0190] NMR (200 MHz, D₂O): δ 8.94 (doublet), δ 8.81 (single) δ 8.63(doublet) 1:1:1. MPt >300° C. λ_(max)(ab) 499,527 nm (water);λ_(max)(em) 552 nm (water).

[0191]4.O-(N-Succinimidyl)-6-{7,14-dioxo-2,9-disulpho-7,14-dihydro-12H-quino[2,3-b]acridin-5-yl}hexanoate

[0192] 4.16-{7,14-Dioxo-2,9-disulpho-7,14-dihydro-12H-quino[2,3-b]acridin-5-yl}hexanoicacid ethyl ester

[0193] Quinacridone-2,9-disulphonic acid (200 mg) was dried by repeatedrotary evaporation with dry DMF. The material was then dissolved in 10ml dry DMSO to give an orange solution. Potassium t-butoxide (45 mg, 0.4mM) was added, the solution immediately turned blue. The solution wasthen stirred magnetically for 30 mins and then ethyl 6-bromohexanoate(70 μl, 0.4 mM) was added and the solution stirred under a nitrogenatmosphere for a further 24 hours. TLC (RP C-18 50/50 methanol/water)showed that approximately half of the starting material had beenconverted to a slightly slower running component. Further reaction timedid not increase the amount of product. Mass spec. showed a peak at 692corresponding to MK₂ ⁺ for the required product. A further peak at 550corresponds to MK₂ ⁺ for the starting material. The solvent was removedby rotary evaporation to leave a red solid. No further attempt was madeto purify this material which was used as such in subsequent reactions.

[0194] 4.26-{7,14-Dioxo-2,9-disulpho-7,14-dihydro-12H-quino[2,3-b]acridin-5-yl}hexanoicacid

[0195] The material from the above reaction was dissolved in 8 ml of amixture of 1M hydrochloric acid: glacial acetic acid (1:3) and heated at100° C. for two hours in a flask fitted with a reflux condenser andunder a nitrogen atmosphere. Mass spec. showed the disappearance of thepeak at 692 as described in the previous example and the appearance of apeak at 664 corresponding to MK₂ ⁺ for6-(7,14-dioxo-2,9-disulpho-7,14-dihydro-12H-quino[2,3-b]acridin-5-yl)hexanoicacid. A further peak at 550 corresponds to MK₂ ⁺ forquinacridone-2,9-disulphonic acid. TLC (as described above showed asingle spot). The solvents were removed by rotary evaporation to give ared solid. No attempt was made to purify this material which was thenused in the next reaction.

[0196] 4.3O-(N-Succinimidyl)-6-{7,14-dioxo-2,9-disulpho-7,14-dihydro-12H-quino[2,3-b]acridin-5-yl}hexanoate

[0197] The material from the previous reaction was dried by repeatedrotary evaporation from DMF. The solid was then dissolved in 10 ml dryDMSO and 200 μl diisopropylethylamine was added followed byO-(succinimidyl)-N,N,N′,N′-tetramethylene uronium hexafluorophosphate(HSPyU, 85 mg). The mixture was stirred at ambient temperature under anitrogen atmosphere for 2 hrs. TLC (as described above) showed thepresence of two spots. Mass spec. showed the disappearance of the peakat 692 as described in the previous example and the appearance of a peakat 684 corresponding to MH⁺ forO-(N-succinimidyl)-6-{7,14-dioxo-2,9-disulpho-7,14-dihydro-12H-quino[2,3-b]acridin-5-yl}hexanoate.A further peak at 550 corresponds to MK₂ ⁺ forquinacridone-2,9-disulphonic acid. The solvent was removed by rotaryevaporation to leave a red gum. Trituration with ethyl acetate gave 74mg of a red solid.

[0198] 5.O-{N-succinimidyl-6-(12-ethyl-7,14-dioxo-2,9-disulpho-7,14-dihydro-12H-quino[2,3-b]acridin-5-yl}hexanoate

[0199] 5.16-{12-ethyl-7,14-dioxo-7,14-dihydro-12H-quino[2,3-b]acridin-5-yl}hexanoicacid, ethyl ester

[0200] Quinacridone (1.56 gm; 5.0 mmol) was suspended in anhydrousdimethylformamide (15 ml) and anhydrous dimethyl sulphoxide (15 ml)under a nitrogen atmosphere. Sodium hydride (60% suspension in oil; 240mg; 6.0 mmol) was added and the mixture stirred until effervescencestopped. More sodium hydride (240 mg; 6.0 mmol) was added and themixture stirred for 10 minutes when effervescence had ceased. Thereaction was heated to 60° C. for 1 hour. Ethyl 6-bromohexanoate (890μl; 5.0 mmol) was added to the dark green solution and the mixturestirred overnight at 60° C. Iodoethane (1.0 ml; 12.5 mmol) was thenadded and the mixture stirred for 2 hours at 60° C. The dark orange-redsolution was allowed to cool, then the mixture was poured into water(300 ml). The solid was filtered off, washed with water and air dried.The solid was then dissolved in dichloromethane (300 ml) and anhydrousmagnesium sulphate added. The mixture was filtered and the solventremoved by rotary evaporation to give a red solid. This was purified byflash chromatography (silica, 15% ethyl acetate/dichloromethane) to give1.04 gm (43%) of the diethyl ester of6-{12-ethyl-7,14-dioxo-7,14-dihydro-12H-quino[2,3-b]acridin-5-yl}hexanoicacid.

[0201] 5.26-{12-ethyl-7,14-dioxo-2,9-disulpho-7,14-dihydro-12H-quino[2,3-b]acridin-5-yl}hexanoicacid

[0202] The diethyl ester of6-{12-ethyl-7,14-dioxo-7,14-dihydro-12H-quino[2,3-b]acridin-5-yl}hexanoicacid (241 mg; 0.5 mmol) was dissolved in conc. sulphuric acid (5 ml) andthe purple solution heated at 110° C. overnight under an atmosphere ofnitrogen. The reaction was allowed to cool and then poured onto ice (˜20gm). The solution was neutralised with 40% w/v sodium hydroxide solutionto give a bright red solution. This was acidified with glacial aceticacid when a orange-red precipitate formed. This was collected bycentrifugation, then dissolved in 0.1% trifluoroacetic acid (TFA) inwater. The solution was purified by reverse phase HPLC. Vydac C18semi-preparative column, water to acetonitrile gradient (both containing0.1% v/v TFA), flow 5 ml/minute, detection at 530 nm. Purified materialwas pooled, evaporated to dryness under vacuum and then dried undervacuum over phosphorous pentoxide to give 300 mg (97%) of6-{12-ethyl-7,14-dioxo-2,9-disulpho-7,14-dihydro-12H-quino[2,3-b]acridin-5-yl}hexanoicacid as a dark red solid.

[0203] δ_(H) (200 MHz, CD₃OD +D₂O) 1.48(3H, t), 1.8(6H, m), 2.38(2H, m),4.52(4H, m), 7.59(2H, m), 8.10(2H, d), 8.60(2H, m), 8.89(2H, d).

[0204] Accurate mass: (M+H)=C₂₈H₂₇N₂O₁₀S₂, requires 615.1107. Found615.1089 (2.9 ppm).

[0205] λ_(max)(ab) 294 nm (ε=65,800/M⁻¹cm⁻¹); 506 nm (εs=5590/M⁻¹cm⁻¹);536 nm.

[0206] (ε=4790/M⁻¹cm⁻¹). (PBS buffer).

[0207] λ_(max)(em) 563 nm (PBS buffer).

[0208] 5.3O-{N-Succinimidyl-6-(12-ethyl-7,14-dioxo-2,9-disulpho-7,14-dihydro-12H-quino[2,3-b]acridin-5-yl}hexanoate.

[0209]6-{12-Ethyl-7,14-dioxo-2,9-disulpho-7,14-dihydro-12H-quino[2,3-b]acridin-5-yl}hexanoicacid (15 mg; 241 μmol), O-(N-succinimidyl)-N,N,N′,N′-tetramethyluroniumtetrafluoroborate (8 mg; 27 μmol), anhydrous dimethyl sulphoxide (500μl) and diisopropylethylamine (17.5 μl) were mixed to give an orangesolution. This was left for 30 minutes when TLC (RP₁₈ 30:70water:methanol) showed that the starting material had been converted toa slower running component identified asO-(N-succinimidyl-6-{12-ethyl-7,14-dioxo-2,9-disulpho-7,14-dihydro-12H-quino[2,3-b]acridin-5-yl}hexanoateby mass spectroscopy.

[0210] Mass spectrum: (ES+) (M+H) 712.

[0211] 6.6-{2,9-Dibromo-12-(5-carboxypentyl)-7,14-dioxo-7,14-dihydro-12H-quino[2,3-b]acridin-5-yl}hexanoicacid

[0212] 6.1 Dimethyl2,5-bis{(4-bromophenyl)amino}cyclohexa-1,4-diene-1,4-dicarboxylate

[0213] Dimethyl 1,4-cyclohexanedione-2,5-dicarboxylate (4.56 gm; 20mmol) and methanol (100 ml) were heated to boiling, then 4-bromoaniline(6.88gm; 40 mmol) was added followed by conc. hydrochloric acid (200μl). The mixture was refluxed for 5 hours under a nitrogen atmosphere.On cooling a cream solid precipitated out which was collected byfiltration, washed with methanol and dried under vacuum to give 10.18 gm(95%) of dimethyl2,5-bis{(4-bromophenyl)amino}cyclohexa-1,4-diene-1,4-dicaboxylate.

[0214] 6.2 2,5-Bis{(4-bromophenyl)amino}terephthalic acid

[0215] Dimethyl2,5-bis{(4-bromophenyl}amino]cyclohexa-1,4-diene-1,4-dicarboxylate (5.36gm; 10 mmol) the sodium salt of 3-nitrobenzenesulphonic acid (2.3 gm; 10mmol), ethanol (50 ml) and 1.0M sodium hydroxide (30 ml) were heated toreflux for 7 hours under a nitrogen atmosphere. The bright yellowsolution was allowed to cool and water (120 ml) was added. The mixturewas acidified with conc. hydrochloric acid when a magenta solidprecipitated out. This material was filtered off, washed with water anddried under vacuum over phosphorous pentoxide to give 4.84 gm (96%) of2,5-bis{(4-bromophenyl)amino}terephthalic acid.

[0216] 6.3 2,9-Dibromoquinacridone

[0217] 2,5-Bis{(4-bromophenyl)amino}terephthalic acid (4.0 gm; 7.9 mmol)and polyphosphoric acid (34 gm) were heated at 150° C. for 4 hours undera nitrogen atmosphere. The mixture was allowed to cool and then pouredinto iced water (100 ml) when a magenta solid was formed. The solid wasfiltered off, washed with water, then methanol and dried under vacuumover phosphorous pentoxide to give 3.68 gm (99%)2,9-dibromoquinacridone.

[0218] 6.46-{2,9-dibromo-12-(5-carboxypentyl)-7,14-dioxo-7,14-dihydro-12H-quino[2,3-b]acridin-5-yl}hexanoicacid, diethyl ester

[0219] 2,9-Dibromoquinacridone (2.35 gm; 5.0 mmol) was suspended inanhydrous dimethylformamide (15 ml) under a nitrogen atmosphere. Sodiumhydride (60% suspension in oil; 480 mg; 12 mmol) was added and themixture stirred until effervescence stopped. Anhydrous dimethylsulphoxide (25 ml) was added. The reaction was heated to 70° C. for 2hour. Ethyl 6-bromohexanoate (2.67 ml; 15 mmol) was added to the darkgreen solution and the mixture stirred overnight at 50° C. The dark bluesolution was allowed to cool, then the mixture was poured into water(200 ml) and acidified with conc. hydrochloric acid. The solid wasfiltered off, washed with water and air dried. This was purified byflash chromatography (silica. 5-20% ethyl acetateldichloromethane) togive 1.74 gm (46%) of6-{2,9-dibromo-12-(5-carboxypentyl)-7,14-dioxo-7,14-dihydro-12H-quino[2,3-b]acridin-5-yl}hexanoicacid, diethyl ester.

[0220] δ_(H) (200 MHz, CDCl₃) 1.25(6H, t), 1.80(12H, m), 2.39(4H, t),4.15(4H, dd), 4.39(4H, t), 7.24(2H, d), 7.70(2H, dd), 8.42(4H, s).

[0221] λ_(max)(ab) 493 nm, 527 nm λ_(max)(em) 560 nm, 600 nm.

[0222] (Dichloromethane).

[0223] 6.56-{2,9-dibromo-12-(5-carboxypentyl)-7,14-dioxo-7,14-dihydro-12H-quino[2,3-b]acridin-5-yl}hexanoicacid

[0224]6-{2,9-Dibromo-12-(5-carboxypentyl)-7,14-dioxo-7,14-dihydro-12H-quino[2,3-b]acridin-5-yl}hexanoicacid, diethyl ester (1.0 gm) was dissolved in glacial acetic acid (20ml) to give a deep magenta solution. 1.0M hydrochloric acid (10 ml) wasadded and the mixture heated to reflux for 5 hours. The reaction wasallowed to cool, the red precipitate filtered off, washed with aceticacid and then diethyl ether and dried under vacuum over phosphorouspentoxide to give 0.86 gm (93%) of6-{2,9-dibromo-12-(5-carboxypentyl)-7,14-dioxo-7,14-dihydro-12H-quino[2,3-b]acridin-5-yl}hexanoicacid.

[0225] Mass spectrum: (ES+) (M+H) 753, 755, 757.

[0226] λ_(max)(ab) 499 nm, 533 nm. λ_(max)(em) 552 nm, 595 nm.(methanol).

[0227] 7.6-{2,9-Dichloro-12-(5-carboxypentyI)-7,14-dioxo-7,14-dihydro-12H-quino[2,3-b]acridin-5-yl}hexanoicacid, diethyl ester

[0228] 7.1 Dimethyl2,5-bis{(4-chlorophenyl)amino}cyclohexa-1,4-diene-1,4-dicarboxylate

[0229] Dimethyl 1,4-cyclohexanedione-2,5-dicarboxylate (4.5 6 gm; 20mmol) and methanol (100 ml) were heated to boiling; then 4-chloroaniline(5.36 gm; 42 mmol) was added followed by conc. hydrochloric acid (200μl). The mixture was refluxed for 5 hours under a nitrogen atmosphere.On cooling, a cream solid precipitated out which was collected byfiltration, washed with methanol and dried under vacuum to give 8.62 gm(96%) of dimethyl2,5-bis{(4-chlorophenyl)amino}cyclohexa-1,4-diene-1,4-dicarboxylate.

[0230] 7.2 2,5-Bis{(4-chlorophenyl)amino}terephthalic acid

[0231] Dimethyl2,5-bis{(4-chlorophenyl)amino}cyclohexa-1,4-diene-1,4-dicarboxylate(4.47 gm, 10 mmol), the sodium salt of 3-nitrobenzenesulphonic acid (2.3gm; 10 mmol), ethanol (70 ml) and 1.0M sodium hydroxide (40 ml) wereheated to reflux overnight under a nitrogen atmosphere. The brightyellow solution was allowed to cool and water (120 ml) was added. Themixture was acidified with conc. hydrochloric acid when a red solidprecipitated out. This material was filtered off, washed with water anddried under vacuum over phosphorous pentoxide to give 4.0 gm (96%) of2,5-bis{(4-chlorophenyl)amino}terephthalic acid.

[0232] λ_(max)(ab) 308 nm, 379 nm. (0.1M sodium hydroxide).

[0233] Mass spectrum (ES+) (M+H) 417.

[0234] 7.3 2,9-Dichloroquinacridone

[0235] 2,5-Bis{(4-chlorophenyl)amino}terephthalic acid (3.35 gm; 8 mmol)and polyphosphoric acid (30 gm) were heated at 150° C. for 3 hours undera nitrogen atmosphere. The mixture was allowed to cool and then pouredinto iced water (200 ml) when a magenta solid precipitated out. This wasfiltered off, washed with water and methanol, then dried under vacuumover phosphorous pentoxide to give 3.1 gm (100%) of2,9-dichloroquinacridone.

[0236] Mass spectrum (ES+) (M+H) 381.

[0237] 7.46-{2,9-Dichloro-12-(5-carboxypentyl)-7,14-dioxo-7,14-dihydro-12H-quino[2,3-b]acridin-5-yl}hexanoicacid, diethyl ester

[0238] 2,9-Dichloroquinacridone (381 mg; 1.0 mmol) was suspended inanhydrous dimethylformamide (4 ml) under a nitrogen atmosphere. Sodiumhydride (60% suspension in oil; 100 mg; 2.40 mmol) was added and themixture stirred until effervescence stopped. Anhydrous dimethylsulphoxide (7 ml) was added. The reaction was heated to 70° C. for 1hour. Ethyl 6-bromohexanoate (535 μl; 3.0 mmol) was added to the darkgreen solution and the mixture stirred overnight at 70° C. The darkorange-red solution was allowed to cool, then the mixture was pouredinto water (150 ml) and 1.0M hydrochloric acid (10 ml). The solid wasfiltered off, washed with water and air dried. This was purified byflash chromatography (silica. 20% ethyl acetate/dichloromethane) to givea red oil which crystallised on triturating with diethyl ether to give205 mg (31%) of6-{2,9-dichloro-12-(5-carboxy-pentyl)-7,14-dioxo-7,14-dihydro-12H-quino[2,3-b]acridin-5-yl}hexanoicacid, diethyl ester.

[0239] Mass spectrum (ES+) (M+H) 665.

[0240] δ_(H) (200 MHz, CDCl₃) 1.27(6H, t), 1.80(12H, m), 2.39(4H, t),4.15(4H, dd), 4.46(4H, t), 7.40(2H, d). 7.64(2H, dd), 8.40(2H, d),8.6(2H, s).

[0241] λ_(max)(ab) 464 nm, 493 nm, 528 nm. λ_(max)(em) 560 nm, 600 nm.

[0242] (Dichloromethane).

[0243] 8.6-{2,9-Difluoro-12-(5-carboxypentyl)-7,14-dioxo-7,14-dihydro-12H-quino[2,3-b]acridin-5-yl}hexanoicacid, diethyl ester

[0244] 8.1 Dimethyl2,5-bis{(4-fluorophenyl)amino}cyclohexa-1,4-diene-1,4-dicarboxylate

[0245] Dimethyl 1,4-cyclohexanedione-2,5-dicarboxylate (9.12 gm; 40mmol) and methanol (200 ml) were heated to boiling, then 4-fluoroaniline(8.35 ml (9.78 gm); 42 mmol) was added followed by conc. hydrochloricacid (400 μl). The mixture was refluxed for 3 hours under a nitrogenatmosphere. On cooling a yellow solid precipitated out which wascollected by filtration, washed with methanol and dried under vacuum togive 15.8 gm (96%) of dimethyl2,5-bis{(4-fluorophenyl)amino}cyclohexa-1,4-diene-1,4-dicarboxylate.

[0246] 8.2 2,5-Bis{(4-fluorophenyl)amino}terephthalic acid

[0247] Dimethyl2,5-bis{(4-fluorophenyl)amino}cyclohexa-1,4-diene-1,4-dicarboxylate(6.21 gm, 15 mmol), the sodium salt of 3-nitrobenzenesulphonic acid (3.6gm; 16 mmol), ethanol (90 ml) and 1.0M sodium hydroxide (50 μl) wereheated to reflux overnight under a nitrogen atmosphere. The brightyellow solution was allowed to cool and water (120 ml) was added. Themixture was acidified with conc. hydrochloric acid when a red solidprecipitated out. This material was filtered off, washed with water anddried under vacuum over phosphorous pentoxide to give 5.6 gm (97%) of2,5-bis{(4-fluorophenyl)amino}terephthalic acid λ_(max)(ab) 295 nm, 380nm. (0.1M sodium hydroxide)

[0248] 8.3 2,9-Difluoroquinacridone

[0249] 2,5-Bis{(4-fluorophenyl)amino}terephthalic acid (5.0 gm; 13 mmol)and polyphosphoric acid (˜50 gm) were heated at 150° C. for 3 hoursunder a nitrogen atmosphere. The mixture was allowed to cool and thenpoured into iced water (200 ml) when a magenta solid precipitated out.This was filtered off, washed with water and then methanol, then driedunder vacuum over phosphorous pentoxide to give 4.5 gm (99%) of2,9-difluoroquinacridone.

[0250] Mass spectrum (ES+) (M+H) 349

[0251] 8.46-{2,9-Difluoro-12-(5-carboxypentyl)-7,14-dioxo-7,14-dihydro-12H-quino[2,3-b]acridin-5-yl}hexanoicacid, diethyl ester

[0252] 2,9-Difluoroquinacridone (350 mg; 1.0 mmol) was suspended inanhydrous dimethylformamide,(4 ml) under a nitrogen atmosphere. Sodiumhydride (60% suspension in oil; 100 mg; 2.40 mmol) was added and themixture stirred until effervescence stopped. The reaction was heated to70° C. for 1 hour. Ethyl 6-bromohexanoate (535 μl; 3.0 mmol) was addedto the dark green solution and the mixture stirred overnight at 70° C.The dark orange-red solution was allowed to cool; then the mixture waspoured into water (150 ml) and 1.0M hydrochloric acid. The solid wasfiltered off, washed with water and air dried. This was purified byflash chromatography (silica. 20% ethyl acetate/dichloromethane) to givea red oil which crystallised on triturating with diethyl ether to give171 mg (27%) of6-{2,9-difluoro-12-(5-carboxypentyl)-7,14-dioxo-7,14-dihydro-12H-quino[2,3-b]acridin-5-yl}hexanoicacid, diethyl ester.

[0253] δ_(H) (200 MHz, CDCl₃) 1.27(6H, t), 1.80(12H, m), 2.39(4H, t),4.15(4H, dd), 4.48(4H, t), 7.46(4H, dd), 8.12(2H, d), 8.61(2H, s).

[0254] λ_(max)(ab) 495 nm, 533 nm. λ_(max)(em) 570 nm, 605 nm.

[0255] (Dichloromethane).

[0256] Mass spectrum (ES+) (M+H) 633.

[0257] 9.6-{2,9-dimethyl-12-(5-carboxypentyl)-7,14-dioxo-7,14-dihydro-12H-quino[2,3-b]acridin-5-yl}hexanoicacid, diethyl ester

[0258] 9.1Dimethyl2,5-bis{(4-methylphenyl)amino}cyclohexa-1,4-diene-1,4-dicarboxylate

[0259] Dimethyl 1,4-cyclohexanedione-2,5-dicarboxylate (4.56 gm; 20mmol) and methanol (100 ml) were heated to boiling; then 4-methylaniline(4.5 gm; 42 mmol) was added followed by conc. hydrochloric acid (200μl). The mixture was refluxed for 5 hours under a nitrogen atmosphere.On cooling a cream solid precipitated out which was collected byfiltration, washed with methanol and dried under vacuum to give 7.92 gm(97%) of dimethyl2,5-bis{(4-methylphenyl)amino}cyclohexa-1,4-diene-1,4-dicarboxylate.

[0260] 9.2 2,5-Bis{(4-methylphenyl)amino}terephthalic acid

[0261] Dimethyl2,5-bis{(4-methylphenyl)amino}cyclohexa-1,4-diene-1,4-dicarboxylate (5.1gm, 12.5 mmol), sodium salt of 3-nitrobenzenesulphonic acid (2.93 gm; 13mmol), ethanol (75 ml) and 1.0M sodium hydroxide (40 ml) were heated toreflux overnight under a nitrogen atmosphere. The bright yellow solutionwas allowed to cool and water (120 ml) was added. The mixture wasacidified with conc. hydrochloric acid when a purple solid precipitatedout. This material was filtered off, washed with water and dried undervacuum over phosphorous pentoxide to give 4.53 gm is (96%) of2,5-bis{(4-chlorophenyl)amino}terephthalic acid.

[0262] λ_(max)(ab) 299 nm, 386 nm. (0.1M sodium hydroxide).

[0263] 9.3 2,9-Dimethylquinacridone

[0264] 2,5-Bis{(4-methylphenyl)amino}terephthalic acid (3.76 gm; 10mmol) and polyphosphoric acid (40 gm) were heated at 150° C. for 2.5hours under a nitrogen atmosphere. The mixture was allowed to cool andthen poured into iced water (100 ml) when a magenta solid precipitatedout. This was filtered off, washed with water and then methanol, thendried under vacuum over phosphorous pentoxide to give 3.12 gm (92%) of2,9-dimethylquinacridone.

[0265] 9.46-{2,9-Dimethyl-12-(5-carboxypentyl)-7,14-dioxo-7,14-dihydro-12H-quino[2,3-b]acridin-5-yl}hexanoicacid, diethyl ester

[0266] 2,9-Dimethylquinacridone (680 mg; 2.0 mmol) was suspended in amixture of anhydrous dimethylformamide (10 ml) and anhydrous dimethylsulphoxide (10 ml) under a nitrogen atmosphere. Sodium hydride (60%suspension in oil; 200 mg; 5.0 mmol) was added and the mixture stirreduntil effervescence stopped. The reaction was heated to 70° C. for 1hour. Ethyl 6-bromohexanoate (1.07 ml (1.34 gm); 6.0 mmol) was added tothe dark blue-green solution and the mixture stirred overnight at 60° C.The dark orange-red solution was allowed to cool, then the mixture waspoured into water (150 ml) and 1.0M hydrochloric acid (30 ml). The solidwas filtered off, washed with water and air dried. This was purified byflash chromatography (silica, 5-25% ethyl acetate/dichloromethane) togive a red oil which crystallised on triturating with diethyl ether togive 780 mg (62%) of6-{2,9-dimethyl-12-(5-carboxypentyl)-7,14-dioxo-7,14-dihydro-12H-quino[2,3-b]acridin-5-yl}hexanoicacid, diethyl ester.

[0267] δ_(H) (200 MHz, CDCl₃) 1.24 (6H, t), 1.80(12H, m), 2.38(10H, m),4.15(4H, dd), 4.45(4H, t), 7.30(2H dd), 7.47(2H, dd), 8.21(2H, d),8.57(2H, s).

[0268] λ_(max)(ab) 495 nm, 530 nm λ_(max)(em) 545 nm. (Dichloromethane).

[0269] 10.6-{2,9-Dimethoxy-12-(5-carboxypentyl)-7,14-dioxo-7,14-dihydro-12H-quino[2,3-b]acridin-5-yl}hexanoicacid, diethyl ester

[0270] 10.1 Dimethyl2,5-bis{(4-methoxyphenyl)amino}cyclohexa-1,4-diene-1,4-dicarboxylate

[0271] Dimethyl 1,4-cyclohexanedione-2,5-dicarboxylate (9.12 gm; 40mmol) and methanol (200 ml) were heated to boiling, then4-methoxyaniline (10.84 gm; 88 mmol) was added followed by conc.hydrochloric acid (400 μl). The mixture was refluxed for 2.5 hours undera nitrogen atmosphere. On cooling, an orange solid precipitated outwhich was collected by filtration, washed with methanol and dried undervacuum to give 17.0 gm (97%) of dimethyl2,5-bis{(4-methoxyphenyl)amino}cyclohexa-1,4-diene-1,4-dicarboxylate.

[0272] 10.2 2,5-Bis{(4-methoxyphenyl)amino}terephthalic acid

[0273] Dimethyl2,5-bis{(4-methoxyphenyl)amino}cyclohexa-1,4-diene-1,4-dicarboxylate(6.58 gm, 15 mmol), the sodium salt of 3-nitrobenzenesulphonic acid (3.6gm; 16 mmol), ethanol (90 ml) and 1.0M sodium hydroxide (50 ml) wereheated to reflux overnight under a nitrogen atmosphere. The orangesolution was allowed to cool and water (120 ml) was added. The mixturewas acidified with conc. hydrochloric acid when a purple solidprecipitated out. This material was filtered off, washed with water,then 25% ethanol/water and dried under vacuum over phosphorous pentoxideto give 6.0 gm (98%) of 2,5-bis{(4-methoxyphenyl)amino}terephthalicacid.

[0274] λ_(max)(ab) 299 nm, 392 nm. (0.1M sodium hydroxide).

[0275] 10.3 2,9-Dimethoxyquinacridone

[0276] 2,5-Bis{(4-methoxyphenyl)amino}terephthalic acid (1.02 gm; 2.5mmol) and polyphosphoric acid (10 gm) were heated at 160° C. for 15minutes under a nitrogen atmosphere. The mixture was allowed to cool andthen poured into iced water (200 ml) when a purple solid precipitatedout. This was filtered off, washed with water and methanol, then driedunder vacuum over phosphorous pentoxide to give 948 mg (100%) of2,9-dimethoxyquinacridone.

[0277] Mass spectrum (ES+) (M+H) 373.

[0278] 10.4 Diethyl ester of6-{2,9-dimethoxy-12-(5-carboxypentyl)-7,14-dioxo-7,14-dihydro-12H-quino[2,3-b]acridin-5-yl}hexanoicacid.

[0279] 2,9-Dimethoxyquinacridone (375 mg; 1.0 mmol) was suspended in amixture of anhydrous dimethylformamide (5 ml) and anhydrous dimethylsulphoxide (5 ml) under a nitrogen atmosphere. Sodium hydride (60%suspension in oil; 100 mg; 2.40 mmol) was added and the mixture stirreduntil effervescence stopped. The reaction was heated to 70° C. for 1hour. Ethyl 6-bromohexanoate (535 μl; 3.0 mmol) was added to the darkgreen solution and the mixture stirred overnight at 70° C. The darkpurple-red solution was allowed to cool, then the mixture was pouredinto water (150 ml) and 1.0M hydrochloric acid (20 ml). The solid wasfiltered off, washed with water and air dried. This was purified byflash chromatography (silica. 5-30% ethyl acetate/dichloromethane) togive 230 mg (35%) of6-{2,9-dimethoxy-12-(5-carboxypentyl)-7,14-dioxo-7,14-dihydro-12H-quino[2,3-b]acridin-5-yl}hexanoicacid, diethyl ester as a red solid. δ_(H) (200 MHz, CDCl₃) 1.25(6H, t),1.80(12H, m), 2.40(4H, t), 4.00(6H, s), 4.15(4H, m), 4.50(4H, t ),7.42(4H, m), 7.91(2H, d), 8.70(2H, s). λ_(max)(ab) 510 nm, 547 nm.λ_(max)(em) 592 nm. (methanol).

[0280] Mass spectrum (ES+) (M+H) 656 (M+Na) 679.

[0281] 11.6-{2,9-Dinitro-12-(5-carboxypentyl)-7,14-dioxo-7,14-dihydro-12H-quino[2,3-b]acridin-5-yl}hexanoicacid, diethyl ester

[0282]5,12-Bis(O-ethyl-6-hexanoyl)-5,12-dihydroquino[2,3-b]acridin-7,14-dione(300 mg; 0.5 mmol) was cooled in an ice bath and then dissolved in conc.sulphuric acid (3 ml) under a nitrogen atmosphere to give a purplesolution. Conc. nitric acid (70 μl; 1.08 mmol) was added and thereaction mix removed from the ice bath. After one hour, the reaction mixwas added to ice when an orange precipitate formed. The mixture wasextracted with dichloromethane. The organic phase was washed with dilutesodium bicarbonate solution, then dried with anhydrous magnesiumsulphate. After filtration, the solvent was removed by rotaryevaporation to give an orange solid. This was purified by flashchromatography (silica. 2-3% methanol/dichloromethane). After removal ofsolvent, the residue was triturated with diethyl ether to give 240 mg(70%) of6-{2,9-dinitro-12-(5-carboxypentyl)-7,14-dioxo-7,14-dihydro-12H-quino[2,3-b]acridin-5-yl}hexanoicacid, diethyl ester as an orange solid.

[0283] δ_(H) (200 MHz, CD₃OD) 1.28(6H, t), 1.80(12H, m), 2.43(4H, t),4.17(4H, dd), 4.56(4H, t), 7.56(2H, d), 8.48(2H, dd), 8.66(2H, s),9.30(2H, d).

[0284] λ_(max)(ab) 408 nm, 474 nm, 506 nm. λ_(max)(em) 518 nm, 556 nm.

[0285] (Dichloromethane).

[0286] N.B. This material has very weak or is non-fluorescent in DMF,DMSO and methanol.

[0287] 12. Fluorescence Lifetime Studies

[0288]FIG. 2 is a plot showing the fluorescence lifetimes of certaindyes according to the invention. Fluorescence lifetimes of a range ofdyes were determined by a time-correlated single photon countingtechnique using an Edinburgh Instruments FL900 CDT Time-ResolvedFluorometer. Samples were excited at 500 nm using a hydrogen-filledflashlamp. Detection was at 550 nm. Deconvolution using a non-linearleast-squares algorithm gave the results shown in Table 2. TABLE 2Fluorescence Lifetimes Compound Solvent Lifetime5,12-Di-n-hexylquinacridone CH₂Cl₂/MeOH 26.5 nsec 2,4,9,11-Quinacridonetetrasulphonic acid water 22.1 nsec 2,9-Quinacridone disulphonic acidwater 20.6 nsec 6-(7,14-Dioxo-2,9-disulpho-7,14-dihydro- water 20.1 nsec12H-quino[2,3-b]acridin-5-yl)hexanoic acid Quinacridone DMSO   22 nsec2-Bromo-5,12-di-n-hexylquinacridone CH₂Cl₂/MeOH 20.4 nsec2,9-Dibromo-5,12-di-n-hexylquinacridone CH₂Cl₂/MeOH 16.9 nsec6-(12-Ethyl-7,14-dioxo-2,9-disulpho-7,14- water 22.7 nsecdihydro-12H-quino[2,3-b]acridin-5-yl) hexanoic acid6-{2,9-Dibromo-12-(5-carboxypentyl)-7,14- water 18.0 nsecdioxo-7,14-dihydro-12H-quino[2,3-b]acridin- 5-yl}hexanoic acid, diethylester. 6-{2,9-Dibromo-12-(5-carboxypentyl)-7,14- MeOH/water 20.7 nsecdioxo-7,14-dihydro-12H-quino[2,3-b]acridin- 17.7 nsec 5-yl}hexanoicacid. 6-{2,9-Dichloro-12-(5-carboxypentyl)-7,14- MeOH 22.3 nsecdioxo-7,14-dihydro-12H-quino[2,3-b]acridin- 5-yl}hexanoic acid, diethylester. 6-{2,9-Difluoro-12-(5-carboxypentyl)-7,14- MeOH 21.4 nsecdioxo-7,14-dihydro-12H-quino[2,3-b]acridin- 5-yl}hexanoic acid, diethylester. 6-{2,9-Dimethyl-12-(5-carboxypentyl)-7,14- MeOH 21.9 nsecdioxo-7,14-dihydro-12H-quino[2,3-b]acridin- 5-yl}hexanoic acid, diethylester 6-{2,9-Dimethoxy-12-(5-carboxypentyl)- EtOH 14.0 nsec7,14-dioxo-7,14-dihydro-12H-quino[2,3-b] acridin-5-yl}hexanoic acid,diethyl ester. 6-{2,9-Dinitro-12-(5-carboxypentyl)-7,14- CH₂Cl₂ 17.0nsec dioxo-7,14-dihydro-12H-quino[2,3-b]acridin- 5-yl}hexanoic acid,diethyl ester.

[0289] 13. Protein Labelling

[0290] 13.1 Preparation of a conjugate of6-{7,14-dioxo-2,9-disulpho-7,14-dihydro-12H-quino[2,3-b]acridin-5-yl}hexanoicacid with ovalbumin

[0291] To 10 ml of ovalbumin (1 mg/ml in 0.1M carbonate buffer, pH 9.3)was added 100 μl ofO-(N-succinimidyl)-6-{7,14-dioxo-2,9-disulpho-7,14-dihydro-12H-quino[2,3-b]acridin-5-yl}hexanoicacid (1 mg/100 μl in DMSO) dropwise whilst stirring. Gentle stirringcontinued for 1 hr at ambient temperature in a foil wrapped vial.Unconjugated dye was removed by overnight dialysis (12-14K MWCO) at +4°C. with at least 2 changes of PBS. The dye-conjugate (Conjugate A) wasrecovered and stored at +4° C.

[0292] 13.2 Determination of the Fluorescence Lifetimes of6-{7,14-dioxo-2,9-disulpho-7,14-dihydro-12H-quino[2,3-b]acridin-5-yl}hexanoicacid and its conjugate with ovalbumin (Conjugate A)

[0293] The fluorescence lifetimes of6-{7,14-dioxo-2,9-disulpho-7,14-dihydro-12H-quino[2,3-b]acridin-5-yl}hexanoicacid and its conjugate with ovalbumin (Conjugate A) was determined inPBS. The results are shown in FIG. 3. Deconvolution and curve fittingusing a non-linear least-squares algorithm gave the results shown inTable 3. TABLE 3 Fluorescence Lifetimes Name Lifetime (nsec)6-{7,14-Dioxo-2,9-disulpho-7,14-dihydro-12H- 20.1quino[2,3-b]acridin-5-yl}hexanoic acid6-{7,14-Dioxo-2,9-disulpho-7,14-dihydro-12H- 19.8quino[2,3-b]acridin-5-yl}hexanoic acid - Ovalbumin conjugate (ConjugateA)

[0294] 14. Labelling of MMP3 peptide substrate withO-{N-succinimidyl-6-(12-ethyl-7,14-dioxo-2,9-disulpho-7,14-dihydro-12H-quino[2,3-b]acridin-5-yl}hexanoate

[0295] The MMP3 peptide substrate (NH₂-RPKPVE(Nva)WRK-NH₂) wassynthesised on a Applied Biosystems model 431A peptide synthesiser usingstandard Fmoc chemistry and Rink amide resin. At the end of thesynthesis the N-terminal Fmoc group was removed. The partially protectedpeptide was left attached to the solid support, in which form it wasreacted withO-{N-succinimidyl-6-(12-ethyl-7,14-dioxo-2,9-disulpho-7,14-dihydro-12H-quino[2,3-b]acridin-5-yl}hexanoate.The labelled peptide was then cleaved from the solid support usingstandard techniques and then purified by reverse phase HPLC.

[0296] 50 mg of the resin bound peptide (equivalent to 16 μmoles ofpeptide) was weighed into a 1.5 ml screw top polypropylene V-vial towhich was added 12 mg (16 μmoles) ofO-{N-succinimidyl-6-(12-ethyl-7,14-dioxo-2,9-disulpho-7,14-dihydro-12H-quino[2,3-b]acridin-5-yl}hexanoicacid dissolved in 1 ml of anhydrous DMSO followed by 20 μl ofdiisopropylethylamine. The vial was placed on rollers with lightexcluded for 20 hrs at ambient temperature (22° C.). The resin was thenfiltered off using a sintered glass frit, washed with 5 ml dry DMSO, 5ml methanol and finally 5 ml dichloromethane, then dried in vacuo for 2hrs.

[0297] The resin was placed in a small round bottomed flask to which wasadded 2 ml of an ice cold solution of trifluoroacetic acid (1.9 ml),water (50 l) and triisopropylsilane (TIS)(50 μl). The mixture wasstirred magnetically for 90 minutes and allowed to warm to ambienttemperature. The mixture was then filtered through a glass wool plug andallowed to drip into 10 ml of ice cold diethyl ether. The pale yellowprecipitate was spun down, the supernatant removed, the precipitateredissolved in 1 ml trifluoroacetic acid and reprecipitated in 10 ml icecold ether. The precipitate was spun down, washed twice with ether thendried in vacuo.

[0298] The crude labelled peptide was dissolved in water, filteredthrough a 0.45 um Millipore filter and a portion was purified on a25cm×1 cm C-18 Phenomenex Jupiter column (code 00G-4055-N0) using agradient of 0.1% TFA/water to 100% of 0.1% TFA/acetonitrile over 30minutes and a flow of 4 ml/minute. Detection was at 220 and 500 nm. Onemajor peaks was eluted after 13 minutes. The material was freeze driedto give 11.4 mg (6.0 μm) of a red solid.

[0299] Mass spectrum (ES+) (M+H) 1893 (calculated molecular weight ofquinacridone labelled peptide=1892).

[0300] 15. Trypsin Cleavage of a Conjugate of Albumin with6-{12-Ethyl-7,14-dioxo-2,9-disulpho-7,14-dihydro-12H-quino[2,3-b]acridin-5-yl}hexanoicacid Monitored by Fluorescence Polarisation

[0301] 15.1 Preparation of Albumin Conjugate

[0302] To 10 ml of human serum albumin (2 mg/ml in 0.1M carbonatebuffer, pH9.3), was addedO-{N-succinimidyl-6-(12-ethyl-7,14-dioxo-2,9-disulpho-7,14-dihydro-12H-quino[2,3-b]acridin-5-yl}hexanoate(110 μl; 2 mg/ml in DMSO) dropwise whilst stirring. Gentle stirringcontinued for 1 hr at ambient temperature in a foil wrapped vial. ASephadex G25 column (PD10—Amersham Biosciences) was used to purify theconjugate which was eluted in de-ionised water.

[0303] 15.2 Trypsin Cleavage of Albumin Conjugate

[0304] To 10 μl of the conjugate (20 μg) in 2 ml of buffer (20 mM TrispH 7.5; 200 mM NaCl; 6 mM CaCl₂) in a cuvette, either 50 μl buffer (asno enzyme control) or 50 μl (500 μg) of trypsin was added. Measurementof the fluorescence polarisation signal was performed using aFluoroMax-3 spectrofluorometer (JYHoriba), with excitation at 485 nm,and detection at 555 nm for 20 minutes at ambient temperature. Theresults as illustrated in FIG. 4, show that the signal becomes lesspolarised as the albumin conjugate is cleaved into smaller fragments bytrypsin, as expected from polarisation theory.

1. Use of a reagent for labelling a target biological material, whereinsaid reagent is a dye of formula:

wherein: groups R³ and R⁴ are attached to the Z¹ ring structure andgroups R⁵ and R⁶ are attached to the Z² ring structure; Z¹ and Z²independently represent the atoms necessary to complete one ring, twofused ring, or three fused ring aromatic or heteroaromatic systems, eachring having five or six atoms selected from carbon atoms and optionallyno more than two atoms selected from oxygen, nitrogen and sulphur; R³,R⁴, R⁵, R⁶, R⁷ and R⁸ are independently selected from hydrogen, halogen,amide, hydroxyl, cyano, nitro, mono- or di-nitro-substituted benzyl,amino, mono- or di-C₁-C₄ alkyl-substituted amino, sulphydryl, carbonyl,carboxyl, C₁-C₆ alkoxy, acrylate, vinyl, styryl, aryl, heteroaryl,C₁-C₂₀ alkyl, aralkyl, sulphonate, sulphonic acid, quaternary ammonium,the group —E—F and the group —(CH₂—)_(n)Y; R¹ and R² are independentlyselected from hydrogen, mono- or di-nitro-substituted benzyl, C₁-C₂₀alkyl, aralkyl, the group —E—F and the group —(CH₂—)_(n)Y; E is a spacergroup having a chain from 1-60 atoms selected from the group consistingof carbon, nitrogen, oxygen, sulphur and phosphorus atoms and F is atarget bonding group; Y is selected from sulphonate, sulphate,phosphonate, phosphate, quaternary ammonium and carboxyl; and n is aninteger from 1to
 6. 2. Use according to claim 1wherein said dye is afluorescent dye wherein: groups R³ and R⁴ are attached to atoms of theZ¹ ring structure and groups R⁵ and R⁸ are attached to atoms of the Z²ring structure, where Z¹ and Z² are hereinbefore defined; R³, R⁴, R⁵,R⁶, R⁷ and R⁸ are independently selected from hydrogen, halogen, amide,hydroxyl, cyano, amino, mono- or di-C₁-C₄ alkyl-substituted amino,sulphydryl, carbonyl, carboxyl, C₁-C₆ alkoxy, acrylate, vinyl, styryl,aryl, heteroaryl, C₁-C₂₀ alkyl, aralkyl, sulphonate, sulphonic acid,quaternary ammonium, the group —E—F and the group —(CH₂—)_(n)Y; andR¹and R² are independently selected from hydrogen, C₁-C₂₀ alkyl,aralkyl, the group —E—F and the group —(CH₂—)_(n)Y; wherein E, F, Y andn are hereinbefore defined.
 3. Use according to claim 1wherein said dyeis a non-fluorescent or substantially non-fluorescent dye wherein:groups R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, Z¹ and Z² are hereinbeforedefined; and wherein at least one of groups R¹, R², R³, R⁴, R⁵, R⁶, R⁷and R⁸ comprises at least one nitro group.
 4. Use according to claims 1to 3 wherein at least one of groups R¹, R², R³, R⁴, R⁵, R⁶, R⁷ and R⁸ isthe group —E—F where E and F are hereinbefore defined.
 5. Use accordingto any of claims 1 to 4 wherein said target bonding group F comprises areactive group for reacting with a functional group on a targetmaterial, or a functional group for reacting with a reactive group on atarget material.
 6. Use according to claim 5 wherein said reactive groupis selected from carboxyl, succinimidyl ester, sulpho-succinimidylester, isothiocyanate, maleimide, haloacetamide, acid halide, hydrazide,vinylsulphone, dichlorotriazine and phosphoramidite.
 7. Use according toclaim 5 wherein said functional group is selected from hydroxy, amino,sulphydryl, imidazole, carbonyl including aldehyde and ketone, phosphateand thiophosphate.
 8. Use according to any of claims 1 to 7 wherein saidspacer group E is selected from: —(—(CHR′)_(p)——{(CHR′)_(q)—O—(CHR′)_(r)}_(s)— —{(CHR′)_(q)—NR′—(CHR′)_(r)}_(s)——{(CHR′)_(q)—(CH═CH)—(CHR′)_(r)}_(s)— —{(CHR′)_(q)—Ar—(CHR′)_(r)}_(s)——{(CHR′)_(q)—CO—NR′—(CHR′)_(r)}_(s)——{(CHR′)_(q)—CO—Ar—NR′—(CHR′)_(r)}_(s)— where R′ is hydrogen, C₁-C₄alkyl or aryl, which may be optionally substituted with sulphonate, Aris phenylene, optionally substituted with sulphonate, p is 1-20,preferably 1-10, q is 0-10, r is 1-10 and s is 1-5.
 9. A method forlabelling a target biological material the method comprising: i) addingto a liquid containing said target biological material a dye of formula:

wherein: groups R³ and R⁴ are attached to the Z¹ ring structure andgroups R⁵ and R⁶ are attached to the Z² ring structure, where Z¹ and Z²are hereinbefore defined; R³, R⁴, R⁵, R⁶, R⁷ and R⁸ are independentlyselected from hydrogen, halogen, amide, hydroxyl, cyano, amino, mono- ordi-C₁-C₄ alkyl-substituted amino, sulphydryl, carbonyl, carboxyl, C₁-C₆alkoxy, acrylate, vinyl, styryl, aryl, heteroaryl, C₁-C₂₀ alkyl,aralkyl, sulphonate, sulphonic acid, quaternary ammonium, the group —E—Fand the group —(CH₂—)_(n)Y; R¹ and R² are independently selected fromhydrogen, C₁-C₂₀ alkyl, aralkyl, the group —E—F and the group—(CH₂—)_(n)Y; where E, F, Y and n are hereinbefore defined; and ii)incubating said dye with said target biological material underconditions suitable for labelling said target.
 10. A method according toclaim 9 wherein at least one of groups R¹, R², R³, R⁴, R⁵, R⁶, R⁷ and R⁸is the group —E—F where E and F are hereinbefore defined.
 11. A methodaccording to claim 9 or claim 10 wherein said target biological materialis selected from the group consisting of antibody, lipid, protein,peptide, carbohydrate, nucleotides which contain or are derivatized tocontain one or more of an amino, sulphydryl, carbonyl, hydroxyl andcarboxyl, phosphate and thiophosphate groups, and oxy or deoxypolynucleic acids which contain or are derivatized to contain one ormore of an amino, sulphydryl, carbonyl, hydroxyl and carboxyl, phosphateand thiophosphate groups, microbial materials, drugs, hormones, cells,cell membranes and toxins.
 12. A method for the assay of an analyte in asample which method comprises: i) contacting the analyte with a specificbinding partner for said analyte under conditions suitable to cause thebinding of at least a portion of said analyte to said specific bindingpartner to form a complex and wherein one of said analyte and saidspecific binding partner is labelled with a fluorescent dye of formula:

wherein: groups R³ and R⁴ are attached to atoms of the Z¹ ring structureand groups R⁵ and R⁶ are attached to atoms of the Z² ring structure,where Z¹ and Z² are hereinbefore defined; at least one of groups R¹, R²,R³, R⁴, R⁵, R⁶, R⁷ and R⁸ is the group —E—F where E is a spacer grouphaving a chain from 1-60 atoms selected from the group consisting ofcarbon, nitrogen, oxygen, sulphur and phosphorus atoms and F is a targetbonding group; when any of said groups R³, R⁴, R⁵, R⁶, R⁷ and R⁸ is notsaid group —E—F, said remaining groups R³, R⁴, R⁵, R⁶, R⁷ and R⁸ areindependently selected from hydrogen, halogen, amide, hydroxyl, cyano,amino, mono- or di-C₁-C₄ alkyl-substituted amino, sulphydryl, carbonyl,carboxyl, C₁-C₆ alkoxy, acrylate, vinyl, styryl, aryl, heteroaryl,C₁-C₂₀ alkyl, aralkyl, sulphonate, sulphonic acid, quaternary ammoniumand the group —(CH₂—)_(n)Y; and, when any of groups R¹ and R² is notsaid group —E—F, said remaining groups R¹ and R² are independentlyselected from hydrogen, C₁-C₂₀ alkyl, aralkyl and the group—(CH₂—)_(n)Y; wherein Y and n are hereinbefore defined; ii) measuringthe emitted fluorescence of the labelled complex; and iii) correlatingthe emitted fluorescence with the presence or the amount of said analytein said sample.
 13. A method according to claim 12 wherein saidmeasuring step ii) is performed by measuring the fluorescence lifetimeor the fluorescence polarisation of the labelled complex.
 14. A methodaccording to claim 12 or claim 13 wherein said analyte-specific bindingpartners are selected from the group consisting antibodies/antigens,lectins/glycoproteins, biotin/streptavidin, hormone/receptor,enzyme/substrate or co-factor, DNA/DNA, DNA/RNA and DNA/binding protein.15. An assay method for the determination of an enzyme in a sample, themethod comprising: i) providing a substrate for the enzyme wherein thesubstrate is labelled with a fluorescent dye of formula:

wherein: groups R³ and R⁴ are attached to atoms of the Z¹ ring structureand groups R⁵ and R⁶ are attached to atoms of the Z² ring structure,where Z¹ and Z² are hereinbefore defined; at least one of groups R¹, R²,R³, R⁴, R⁵, R⁶, R⁷ and R⁸ is the group —E—F where E is a spacer grouphaving a chain from 1-60 atoms selected from the group consisting ofcarbon, nitrogen, oxygen, sulphur and phosphorus atoms and F is a targetbonding group; when any of said groups R³, R⁴, R⁵, R⁶, R⁷ and R⁸ is notsaid group —E—F, said remaining groups R³, R⁴, R⁵, R⁶, R⁷ and R⁸ areindependently selected from hydrogen, halogen, amide, hydroxyl, cyano,amino, mono- or di-C₁-C₄ alkyl-substituted amino, sulphydryl, carbonyl,carboxyl, C₁-C₆ alkoxy, acrylate, vinyl, styryl, aryl, heteroaryl,C₁-C₂₀ alkyl, aralkyl, sulphonate, sulphonic acid, quaternary ammoniumand the group —(CH₂—)_(n)Y; and, when any of groups R¹ and R² is notsaid group —E—F, said remaining groups R¹ and R² are independentlyselected from hydrogen, C₁-C₂₀ alkyl, aralkyl and the group—(CH₂—)_(n)Y; wherein Y and n are hereinbefore defined; ii) combiningthe labelled substrate with the enzyme under conditions suitable forinitiating the enzymatic reaction; and iii) measuring the fluorescencepolarisation of the sample to determine the extent of reaction.
 16. Aset of two or more different fluorescent dyes, each dye of said set ofdyes having the formula:

wherein: groups R³ and R⁴ are attached to atoms of the Z¹ ring structureand groups R⁵ and R⁶ are attached to atoms of the Z² ring structure,where Z¹ and Z² are hereinbefore defined; R³, R⁴, R⁵, R⁶, R⁷ and R⁸ areindependently selected from hydrogen, halogen, amide, hydroxyl, cyano,amino, mono- or di-C₁-C₄ alkyl-substituted amino, sulphydryl, carbonyl,carboxyl, C₁-C₆ alkoxy, acrylate, vinyl, styryl, aryl, heteroaryl,C₁-C₂₀ alkyl, aralkyl, sulphonate, sulphonic acid, quaternary ammonium,the group —E—F and the group —(CH₂—)_(n)Y; R¹ and R² are independentlyselected from hydrogen, C₁-C₂₀ alkyl, aralkyl, the group —E—F and thegroup —(CH₂—)_(n)Y; E is a spacer group having a chain from 1-60 atomsselected from the group consisting of carbon, nitrogen, oxygen, sulphurand phosphorus atoms and F is a target bonding group; Y is selected fromsulphonate, sulphate, phosphonate, phosphate, quaternary ammonium andcarboxyl; and n is an integer from 1 to 6; wherein each dye of said sethas a distinguishably different fluorescence lifetime compared with thelifetimes of the remaining dyes of the set.
 17. A set according to claim16 wherein at least one of groups R¹, R², R³, R⁴, R⁵, R⁶, R⁷ and R⁸ isthe group —E—F where E and F are hereinbefore defined.
 18. A setaccording to claim 16 or claim 17 comprising four different dyes.
 19. Aset according to any of claims 16 to 18 wherein each dye of said setexhibits a different fluorescence lifetime in the range from 1 to 30nanoseconds, more preferably in the range from 10 to 25 nanoseconds. 20.A set according to any of claims 16 to 19 wherein the lifetime of thefluorescence emission of each of the dyes is separated by at least 0.5nanoseconds.
 21. A method of increasing the fluorescence of anon-fluorescent or substantially non-fluorescent dye of formula:

wherein: groups R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, Z¹ and Z² arehereinbefore defined; and wherein at least one of groups R¹, R², R³, R⁴,R⁵, R⁶, R⁷ and R⁸ comprises at least one nitro group; characterised bythe reduction of said at least one nitro group to —NHOH or —NH₂
 22. Amethod for detecting nitroreductase enzyme activity in a compositioncomprising: i) mixing said composition under conditions to promotenitroreductase activity with a dye of formula:

wherein: groups R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, Z¹ and Z² arehereinbefore defined and wherein at least one of groups R¹, R², R³, R⁴,R⁵, R⁶, R⁷ and R⁸ comprises at least one nitro group; and ii) measuringan increase in fluorescence wherein said increase is a measure of theamount of nitroreductase activity.
 23. An assay method comprising: i)binding one component of a specific binding pair to a surface; ii)adding a second component of the specific binding pair under conditionsto promote binding between the components, said second component beinglabelled with a nitroreductase enzyme; iii) adding a dye of formula:

wherein: groups R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, Z¹ and Z² arehereinbefore defined and wherein at least one of groups R¹, R², R³, R⁴,R⁵, R⁶, R⁷ and R⁸ comprises at least one nitro group; and iv) detectingbinding of the second component to the first component by measuring anincrease in fluorescence as a measure of bound nitroreductase activity.24. A method according to claim 23 wherein said specific binding pair isselected from the group consisting of antibodies/antigens,lectins/glycoproteins, biotin/streptavidin, hormone/receptor,enzyme/substrate, DNA/DNA, DNA/RNA and DNA/binding protein.
 25. An assaymethod which comprises: i) contacting a host cell which has beentransfected with a nucleic acid molecule comprising expression controlsequences operably linked to a sequence encoding a nitroreductase with adye of formula:

wherein: groups R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, Z¹ and Z² arehereinbefore defined and wherein at least one of groups R¹, R², R³, R⁴,R⁵, R⁶, R⁷ and R⁸ comprises at least one nitro group; and ii) measuringan increase in fluorescence as a measure of nitroreductase geneexpression.
 26. A method according to claim 25 wherein said assay isconducted in the presence of a test agent whose effect on geneexpression is to be determined.
 27. A dye of formula:

wherein: groups R³ and R⁴ are attached to atoms of the Z¹ ring structureand groups R⁵ and R⁶ are attached to atoms of the Z² ring structure; Z¹and Z² independently represent the atoms necessary to complete one ring,two fused ring, or three fused ring aromatic or heteroaromatic systems,each ring having five or six atoms selected from carbon atoms andoptionally no more than two atoms selected from oxygen, nitrogen andsulphur; at least one of groups R¹, R², R³, R⁴, R⁵, R⁶, R⁷ and R⁸ is thegroup —E—F where E is a spacer group having a chain from 1-60 atomsselected from the group consisting of carbon, nitrogen, oxygen, sulphurand phosphorus atoms and F is a target bonding group; and, when any ofsaid groups R³, R⁴, R⁵, R⁶, R⁷ and R⁸ is not said group —E—F, saidremaining groups R³, R⁴, R⁵, R⁶, R⁷ and R⁸ are independently selectedfrom hydrogen, halogen, amide, hydroxyl, cyano, nitro, amino, mono- ordi-C₁-C₄ alkyl-substituted amino, sulphydryl, carbonyl, carboxyl, C₁-C₆alkoxy, acrylate, vinyl, styryl, aryl, heteroaryl, C₁-C₂₀ alkyl,aralkyl, sulphonate, sulphonic acid, quaternary ammonium and the group—(CH₂—)_(n)Y; and, when any of groups R¹ and R² is not said group —E—F,said remaining groups R¹ and R² are independently selected fromhydrogen, mono- or di-nitro-substituted benzyl, C₁-C₂₀ alkyl, aralkyland the group —(CH₂—)_(n)Y; E is a spacer group having a chain from 1-60atoms selected from the group consisting of carbon, nitrogen, oxygen,sulphur and phosphorus atoms and F is a target bonding group; Y isselected from sulphonate, sulphate, phosphonate, phosphate, quaternaryammonium and carboxyl; and n is an integer from 1 to 6; provided that atleast one of groups R³, R⁴, R⁵, R⁶, R⁷ and R⁸ is selected fromsulphonate, sulphate, quaternary ammonium and the group —(CH₂—)_(n)Y;and/or at least one of groups R¹ and R² is the group —(CH₂—)_(n)Y, whereY and n are hereinbefore defined.
 28. A dye according to claim 27wherein said target bonding group F comprises: i) a reactive groupselected from carboxyl, succinimidyl ester, sulpho-succinimidyl ester,isothiocyanate, maleimide, haloacetamide, acid halide, hydrazide,vinylsulphone, dichlorotriazine and phosphoramidite; or ii) a functionalgroup selected from hydroxy, amino, sulphydryl, imidazole, carbonylincluding aldehyde and ketone, phosphate and thiophosphate.
 29. A dyeaccording to claim 27 or claim 28 wherein said spacer group E isselected from: —(CHR′)_(p)— —{(CHR′)_(q)—O—(CHR′)_(r)}_(s)——{(CHR′)_(q)—NR′—(CHR′)_(r)}_(s)— —{(CHR′)_(q)—(CH═CH)—(CHR′)_(r)}_(s)——{(CHR′)_(q)—Ar—(CHR′)_(r)}_(s)— —{(CHR′)_(q)—CO—NR′—(CHR′)_(r)}_(s)——{(CHR′)_(q)—CO—Ar—NR′—(CHR′)_(r)}_(s)— where R′ is hydrogen, C₁-C₄alkyl or aryl, which may be optionally substituted with sulphonate, Aris phenylene, optionally substituted with sulphonate, p is 1-20,preferably 1-10, q is 0-10, r is 1-10 and s is 1-5.